Screening and Therapy for Lymphatic Disorders Involving the FLT4 Receptor Tyrosine Kinase (VEGFR-3)

ABSTRACT

The present invention provides materials and methods for screening for and treating hereditary lymphedema in human subjects.

This application is a Continuation-in-Part of International PatentApplication No. PCT/US99/06133, filed Mar. 26, 1999, incorporated hereinby reference in its entirety.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with United States and Finnish governmentsupport, including support under contract R03-HD35174, awarded by theU.S. National Institutes of Health. The U.S. Government has certainrights in this invention.

FIELD OF THE INVENTION

The present invention relates generally to the fields of molecularbiology and medicine; more particularly to the areas of geneticscreening and the identification and treatment of hereditary disorders;and more particularly to identification and treatment of hereditarylymphedema.

DESCRIPTION OF RELATED ART

The lymphatic system is a complex structure organized in parallelfashion to the circulatory system. In contrast to the circulatorysystem, which utilizes the heart to pump blood throughout the body, thelymphatic system pumps lymph fluid using the inherent contractibility ofthe lymphatic vessels. The lymphatic vessels are not interconnected inthe same manner as the blood vessels, but rather form a set ofcoordinated structures including the initial lymphatic sinuses [Jeltschet al., Science, 276:1423-1425 (1997); and Castenholz, A., in Olszewski,W. L. (ed.), Lymph Stasis: Pathophysiology, Diagnosis, and Treatment.CRC Press: Boca Raton, Fla. (1991), pp. 15-42] which drain into thelymphatic capillaries and subsequently to the collecting lymphaticswhich drain into the lymphatic trunks and the thoracic duct whichultimately drains into the venous circulation. The composition of thechannels through which lymph passes is varied [Olszewski, W. L., inOlszewski, W. L. (ed), Lymph Stasis: Pathophysiology, Diagnosis, andTreatment. CRC Press. Boca Raton, Fla. (1991), pp. 235-258, andKinmonth, J. B., in Kinmonth, J. B. (ed), The Lymphatics: Diseases,Lymphography and Surgery. Edward Arnold Publishers: London, England(1972), pp. 82-86], including the single endothelial layers of theinitial lymphatics, the multiple layers of the collecting lymphaticsincluding endothelium, muscular and adventitial layers, and the complexorganization of the lymph node. The various organs of the body such asskin, lung, and GI tract have components of the lymphatics with variousunique features. [See Ohkuma, M., in Olszewski (1991), supra, at pp.157-190; Uhley, H. and Leeds, S., in Olszewski (1991), supra, at pp.191-210; and Barrowman, J. A., in Olszewski (1991), at pp. 221-234).]

Molecular biology has identified at least a few genes and proteinspostulated to have roles mediating the growth and/or embryonicdevelopment of the lymphatic system. One such gene/protein is thereceptor tyrosine kinase designated Flt4 (fms-like tyrosine kinase 4),cloned from human erythroleukaemia cell and placental cDNA libraries.[See U.S. Pat. No. 5,776,755; Aprelikova et al., Cancer Res., 52:746-748 (1992); Galland et al., Genomics, 13: 475-478 (1992); Galland etal, Oncogene, 8: 1233-1240 (1993); and Pajusola et al., Cancer Res.,52:5738-5743 (1992), all incorporated herein by reference.] Studiesshowed that, in mouse embryos, a targeted disruption of the Flt4 geneleads to a failure of the remodeling of the primary vascular network,and death after embryonic day 9.5 [Dumont et al., Science, 282: 946-949(1998)]. These studies suggested that Flt4 has an essential role in thedevelopment of the embryonic vasculature, before the emergence of thelymphatic vessels. However, additional studies indicated that, duringfurther development, the expression of Flt4 becomes restricted mainly tolymphatic vessels [Kaipainen, et al., Proc. Natl. Acad. Sci. USA, 92:3566-3570 (1995)].

In humans, there are two isoforms of the Flt4 protein, designated asFlt4s (short, Genbank Accession No. X68203) and Flt41 (long, GenbankAccession Nos. X68203 and S66407, SEQ ID NO: 1). The sequence of theseisoforms is largely identical, except for divergence that occurs at thecarboxyl terminus of the receptor as a result of alternative mRNAsplicing at the 3′ end. The C-terminus of the long form contains threetyrosyl residues, and one of them (Y1337 (SEQ ID NO: 2)) serves as anautophosphorylation site in the receptor [Fourier et al., Oncogene, 11:921-931 (1995), and Pajusola., et al., Oncogene, 8: 2931-2937 (1993)].Only the long form is detected in human erythroleukaemia (HEL) and in amegakaryoblastic cell line (the DAMI cells), and the mouse Flt4 gene(Genbank Accession No. L07296) only produces one mRNA transcript,corresponding to Flt41 [Galland et al., Oncogene, 8, 1233-1240 (1993),and Pajusola et al., Cancer Res., 52: 5738-5743 (1992)]. These findingssuggest that the long form of Flt4 may be responsible for most of thebiological properties of this receptor. The Flt4 protein is glycosylatedand proteolytically processed in transfected cells [Pajusola et al,Oncogene, 9: 3545-3555 (1994)]. During this process, the 175 kD form ofthe receptor matures to a 195 kD form, which is subsequently cleavedinto a 125 kD C-terminal fragment, and a 75 kD extracellulardomain-containing fragment, which are linked by disulphide bonding inthe mature receptor.

Two growth factors, named vascular endothelial growth factors C and D(VEGF-C and VEGF-D) due to amino acid sequence similarity toearlier-discovered vascular endothelial growth factor, have been shownto bind and activate the tyrosine phosphorylation of Flt4. [Achen etal., Proc. Natl. Acad. Sci. USA, 95: 548-553 (1998); Joukov et al,EMBOJ., 16: 3898-3911; and Joukov et at, EMBO J., 15: 290-298 (1996)].Because of Flt4's growth factor binding properties and the fact thatFlt4 possesses amino acid sequence similarity to two previouslyidentified VEGF receptors (Flt1/VEGFR-1 and KDR/VEGFR-2), Flt4 has alsobeen designated VEGFR-3, and these terms are used interchangeablyherein.

When VEGF-C was intentionally over-expressed under a basal keratinpromoter in transgenic mice, a hyperplastic lymphatic vessel network inthe skin was observed. [Jeltsch et al., Science, 276:1423-1425 (1997).]The results of this study, when combined with the expression pattern ofVEGFR-3 in the lymphatic vasculature, suggest that lymphatic growth maybe induced by VEGF-C and mediated via VECFR-3. Notwithstanding theforegoing insights involving one cell surface receptor and the twoapparent ligands therefor, little is known about the developmentalregulation of the lymphatic system.

Hereditary or primary lymphedema, first described by Milroy in 1892[Milroy, N.Y. Med. J., 56:505-508 (1892)], is a developmental disorderof the lymphatic system which leads to a disabling and disfiguringswelling of the extremities, Hereditary lymphedema generally shows anautosomal dominant pattern of inheritance with reduced penetrance,variable expression, and variable age-at-onset [Greenlee et al.,Lymphology, 26:156-168 (1993)]. Swelling may appear in one or all limbs,varying in degree and distribution. If untreated, such swelling worsensover time. In rare instances angiosarcoma may develop in affectedtissues [Offori et al., Clin. Exp. Dermatol., 18:174-177 (1993)].Despite having been described over a century ago, little progress hasbeen made in understanding the mechanisms causing lymphedema. Along-felt need exists for the identification of the presumed geneticvariations that underlie hereditary lymphedema, to permit betterinformed genetic counseling in affected families, earlier diagnosis andtreatment, and the development of more targeted and effective lymphedematherapeutic regimens. In addition, identification of genetic markers andhigh risk members of lymphedema families facilitates the identificationand management of environmental factors that influence the expressionand severity of a lymphedema phenotype.

SUMMARY OF THE INVENTION

The present invention provides materials and methods that address one ormore of the long-felt needs identified above by identifying a geneticmarker that correlates and is posited to have a causative role in thedevelopment of hereditary lymphedema. The invention is based in part onthe discovery that, in several families with members afflicted withhereditary lymphedema, the lymphedema phenotype correlates with geneticmarkers localized to chromosome 5q34-q35; and that in at least some suchfamilies, a missense mutation in the VEGFR-3 gene (which maps tochromosome 5q34-q35) exists that appears to behave in a loss-of-functiondominant negative manner to decrease tyrosine kinase signaling of thereceptor. In view of the fact that VEGFR-3 acts as a high affinityreceptor for vascular endothelial growth factor C (VEGF-C), a growthfactor whose effects include modulation of the growth of the lymphaticvascular network, these linkage and biochemical studies provide animportant marker for determining a genetic predisposition for lymphedemain healthy individuals; and for diagnosing hereditary lymphedema insymptomatic individuals. Materials and methods for performing suchgenetic analyses are considered aspects of the present invention.

Thus, the invention provides genetic screening procedures that entailanalyzing a person's genome—in particular their VEGFR-3 alleles—todetermine whether the individual possesses a genetic characteristicfound in other individuals that are considered to be afflicted with. orat risk for, developing hereditary lymphedema.

For example, in one embodiment, the invention provides a method fordetermining a hereditary lymphedema development potential in a humansubject comprising the steps of analyzing the coding sequence of theVEGFR-3 genes from the human subject; and determining hereditarylymphedema development potential in said human subject from theanalyzing step.

In another embodiment, the invention provides a method of screening ahuman subject for an increased risk of developing a lymphatic disorder,comprising the steps of (a) assaying nucleic acid of a human subject todetermine a presence or an absence of a mutation altering the encodedVEGFR-3 amino acid sequence or expression of at least one VEGFR-3allele; and (b) screening for an increased risk of developing alymphatic disorder from the presence or absence of said mutation.

By “human subject” is meant any human being, human embryo, or humanfetus. It will be apparent that methods of the present invention will beof particular interest to individuals that have themselves beendiagnosed with lymphedema or have relatives that have been diagnosedwith lymphedema.

By “screening for an increased risk” is meant determination of whether agenetic variation exists in the human subject that correlates with agreater likelihood of developing lymphedema than exists for the humanpopulation as a whole, or for a relevant racial or ethnic humansub-population to which the individual belongs. Both positive andnegative determinations (i.e., determinations that a geneticpredisposition marker is present or is absent) are intended to fallwithin the scope of screening methods of the invention. In preferredembodiments, the presence of a mutation altering the sequence orexpression of at least one Flt4 receptor tyrosine kinase allele in thenucleic acid is correlated with an increased risk of developing alymphatic disorder, whereas the absence of such a mutation is reportedas a negative determination.

By “lymphatic disorder” is meant any clinical condition affecting thelymphatic system, including but not limited to lymphedemas,lymphangiomas, lymphangiosarcomas, lymphangiomatosis, lymphangiectasis,and cystic hygroma. Preferred embodiments are methods of screening ahuman subject for an increased risk of developing a lymphedema disorder,i.e., any disorder that physicians would diagnose as lymphedema and thatis characterized by swelling associated with lymph accumulation, otherthan lymphedemas for which non-genetic causes (e.g., parasites, surgery)are known. By way of example, lymphedema disorders include Milroy-Nonne(OMIM 153100) syndrome-early onset lymphedema [Milroy, N.Y. Med. J.,56:505-508 (1892); and Dale, J. Med. Genet., 22: 474-778 (1985)] andlymphedema praecox (Meige syndrome, OMIM 153200)-late onset lymphedema[Lewis et al., J. Ped., 104:641-648 (1984); Holmes et al., Pediatrics61:575-579 (1978); and Wheeler et al., Plastic Reconstructive Surg.,67:362-364 (1981)] which generally are described as separate entities,both characterized by dominant inheritance. However, there is confusionin the literature about the separation of these disorders. In Milroy'ssyndrome, the presence of edema, which is usually more severe in thelower extremities, is seen from birth. Lymphedema praecox presents in asimilar fashion but the onset of swelling is usually around puberty.Some cases have been reported to develop in the post-pubertal period. Inthe particular analyses described herein, the lymphedema familiesshowing linkage to 5q34-q35 show an early onset for most affectedindividuals, but individuals in these pedigrees have presented during orafter puberty.

The “assaying” step of the invention may involve any techniquesavailable for analyzing nucleic acid to determine its characteristics,including but not limited to well-known techniques such as single-strandconformation polymorphism analysis (SSCP) [Orita et al., Proc Natl.Acad. Sci. USA, 86: 2766-2770 (1989)]; heteroduplex analysis [White etal., Genomics, 12: 301-306 (1992)]; denaturing gradient gelelectrophoresis analysis [Fischer et al., Proc. Natl. Acad. Sci. USA,80: 1579-1583 (1983), and Riesner et al., Electrophoresis, 10: 377-389(1989)]; DNA sequencing; RNase cleavage [Myers et al., Science, 230:1242-1246 (1985)]; chemical cleavage of mismatch techniques [Rowley etal., Genomics, 30: 574-582 (1995); and Roberts et al., Nucl. Acids Res.,25: 3377-3378 (1997)]; restriction fragment length polymorphismanalysis; single nucleotide primer extension analysis [Shumaker et al.,Hum. Mutat., 7: 346-354 (1996); and Pastinen et al., Genome Res., 7:606-614 (1997)]; 5′ nucleus assays [Pease et al., Proc. Natl. Acad. Sci.USA, 91:5022-5026 (1994)], DNA Microchip analysis [Ramsay, G., NatureBiotechnology,, 16: 40-48 (1999); and Chee et al., U.S Pat. No.5,837,832]; and ligase chain reaction [Whiteley et al., U.S. Pat. No.5,521,065]. [See generally, Schafer and Hawkins, Nature Biotechnology,16: 33-39 (1998).] All of the foregoing documents are herebyincorporated by reference in their entirety.

In one preferred embodiment, the assaying involves sequencing of nucleicacid to determine nucleotide sequence thereof, using any availablesequencing technique. [See, e.g., Sanger et al., Proc. Natl. Acad. Sci.(USA), 74: 5463-5467 (1977) (dideoxy chain termination method),Mirzabekov, TIBTECH, 12: 27-3 (1994) (sequencing by hybridization);Drmanac et al., Nature Biotechnology; 16: 54-58 (1998); U.S. Pat. No.5,202,231; and Science, 260: 1649-1652 (1993) (sequencing byhybridization); Kieleczawa et al., Science, 258: 1787-1791 (1992)(sequencing by primer walking); (Douglas et al., Biotechniques, 14:824-828 (1993) (Direct sequencing of PCR products); and Akane et al.,Biotechniques 16: 238-241 (1994); Maxam and Gilbert, Meth. Enzymol., 65:499-560 (1977) (chemical termination sequencing), all incorporatedherein by reference.] The analysis may entail sequencing of the entireVEGFR-3 gene genomic DNA sequence, or portions thereof; or sequencing ofthe entire VEGFR-3 coding sequence or portions thereof. In somecircumstances, the analysis may involve a determination of whether anindividual possesses a particular VEGFR-3 allelic variant, in which casesequencing of only a small portion of nucleic acid—enough to determinethe sequence of a particular codon characterizing the allelic variant—issufficient. This approach is appropriate, for example, when assaying todetermine whether one family member inherited the same allelic variantthat has been previously characterized for another family member, or,more generally, whether a person's genome contains an allelic variantthat has been previously characterized and correlated with heritablelymphedema. More generally, the sequencing may be focused on thoseportions of the VEGFR-3 sequence that encode a VEGFR-3 kinase domain,since several different and apparently causative mutations in affectedindividuals that have been identified correspond to residues within anintracellular VEGFR-3 kinase domain. Referring to SEQ ID NOs: 1 and 2,the two kinase domains of human wild type VEGFR-3 correspond tonucleotides 2546 to 2848 and 3044 to 3514 of SEQ ID NO: 1, which encoderesidues 843 to 943 and 1009 to 1165 of SEQ ID NO: 2. Such kinasedomains are localized to exons 17-20 and 22-26 in the VEGFR-3 gene, sothe sequencing/analysis may be focused on those exons in particular.Molecular modeling suggests that, within these domains, residues G852,G854, G857, K879, E896, H1035, D1037, N1042, D1055, F1056, G1057, E1084,D1096, and R1159 are of particular importance in comprising or shapingthe catalytic pocket of the VEGFR-3 kinase domains, so the sequencingmay focus on these residues (in addition to residues descried herein forwhich mutations have already been identified).

In a related embodiment, the invention provides PCR primers useful foramplifying particular exon sequences of human VEGFR-3 genomic DNA. TheExamples below identify preferred primers for amplifying Exon 17, Exon22, and Exon 24 sequences, where specific missense mutations describedherein map. In addition, the Examples below describe the Exon-Intronjunctions of human VEGFR-3, which, in combination with the VEGFR-3 cDNAsequence provided herein, permit the manufacture of appropriateoligonucleotide primers for other exons. Any such primers of, e.g., 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, or more nucleotides that are identical or exactlycomplementary to a human VEGFR-3 genomic sequence and that includes oris within 50 nucleotides of a VEGFR-3 exon-intron splice site isintended to be within the scope of the invention.

In another embodiment, the assaying step comprises performing ahybridization assay to determine whether nucleic acid from the humansubject has a nucleotide sequence identical to or different from one ormore reference sequences. In a preferred embodiment, the hybridizationinvolves a determination of whether nucleic acid derived from the humansubject will hybridize with one or more oligonucleotides, wherein theoligonucleotides have nucleotide sequences that correspond identicallyto a portion of the VEGFR-3 gene sequence, preferably the VEGFR-3 codingsequence set forth in SEQ ID NO: 1, or that correspond identicallyexcept for one mismatch, The hybridization conditions are selected todifferentiate between perfect sequence complementarity and imperfectmatches differing by one or more bases. Such hybridization experimentsthereby can provide single nucleotide polymorphism sequence informationabout the nucleic acid from the human subject, by virtue of knowing thesequences of the oligonucleotides used in the experiments.

Several of the techniques outlined above involve an analysis wherein oneperforms a polynucleotide migration assay, e.g., on a polyacrylamideelectrophoresis gel, under denaturing or non-denaturing conditions.Nucleic acid derived from the human subject is subjected to gelelectrophoresis, usually adjacent to one or more reference nucleicacids, such as reference VEGFR-3 sequences having a coding sequenceidentical to all or a portion of SEQ ID NO: 1, or identical except forone known polymorphism. The nucleic acid from the human subject and thereference sequence(s) are subjected to similar chemical or enzymatictreatments and then electrophoresed under conditions whereby thepolynucleotides will show a differential migration pattern, unless theycontain identical sequences. [See generally Ausubel et al. (eds.),Current Protocols in Molecular Biolog, New York: John Wiley & Sons, Inc.(1987-1999); and Sambrook et al., (eds.), Molecular Cloning, ALaboratory Manual, Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory Press (1989), both incorporated herein by reference in theirentirety.]

In the context of assaying, the term “nucleic acid of a human subject”is intended to include nucleic acid obtained directly from the humansubject (e.g., DNA or RNA obtained from a biological sample such as ablood, tissue, or other cell or fluid sample); and also nucleic acidderived from nucleic acid obtained directly from the human subject. Byway of non-limiting examples, well known procedures exist for creatingcDNA that is complementary to RNA derived from a biological sample froma human subject, and for amplifying (e.g., via polymerase chain reaction(PCR)) DNA or RNA derived from a biological sample obtained from a humansubject. Any such derived polynucleotide which retains relevantnucleotide sequence information of the human subject's own DNA/RNA isintended to fall within the definition of “nucleic acid of a humansubject” for the purposes of the present invention.

In the context of assaying, the term “mutation” includes addition,deletion, and/or substitution of one or more nucleotides in the VEGFR-3gene sequence. The invention is demonstrated by way of non-limitingexamples set forth below that identify several mutations in VEGFR-3,including single nucleotide polymorphisms that introduce missensemutations into the VEGFR-3 coding sequence (as compared to the VEGFR-3cDNA sequence set forth in SEQ ID NO: 1) and other polymorphisms thatoccur in introns and that are identifiable via sequencing, restrictionfragment length polymorphism, or other techniques. Example 2 provides anassay to determine whether a VEGFR-3 mutation inhibits VEGFR-3signaling. Additional assays to study both ligand binding and signalingactivities of VEGFR-3 are disclosed, e.g., in U.S. Pat. No. 5,776,755and International Patent Publication No. WO 98/33917, published 06 Aug.1998, both of which are incorporated herein by reference in theirentirety. Evidence that a VEGFR-3 mutation inhibits VEGFR-3 signaling isevidence that the mutation may have a causative role in lymphedamaphenotype. However, even mutations that have no apparent causative rolemay serve as useful markers for heritable lymphedema, provided that theappearance of the mutation correlates reliably with the appearance oflymphedema.

In a related embodiment, the invention provides a method of screeningfor a VEGFR-3 hereditary lymphedema genotype in a human subject,comprising the steps of (a) providing a biological sample comprisingnucleic acid from a human subject; (b) analyzing the nucleic acid forthe presence of a mutation or mutations in a VEGFR-3 allele in thenucleic acid of the human subject, (c) determining a VEGFR-3 genotypefrom said analyzing step; and (d) correlating the presence of a mutationin a VEGFR-3 allele with a hereditary lymphedema genotype. In apreferred embodiment, the biological sample is a cell sample containinghuman cells that contain genomic DNA of the human subject.

Although more time consuming and expensive than methods involvingnucleic acid analysis, the invention also may be practiced by assayingprotein of a human subject to determine the presence or absence of anamino acid sequence variation in VEGFR-3 protein from the human subject.Such protein analyses may be performed, e.g., by fragmenting VEGFR-3protein via chemical or enzymatic methods and sequencing the resultantpeptides; or by Western analyses using an antibody having specificityfor a particular allelic variant of VEGFR-3.

The invention also provides materials that are useful for performingmethods of the invention. For example, the present invention providesoligonucleotides useful as probes in the many analyzing techniquesdescribed above. In general, such oligonucleotide probes comprise 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, or 50 nucleotides that have a sequence that isidentical, or exactly complementary, to a portion of a human VEGFR-3gene sequence, or that is identical or exactly complementary except forone nucleotide substitution. In a preferred embodiment, theoligonucleotides have a sequence that corresponds in the foregoingmanner to a human VEGFR-3 coding sequence, and in particular, theVEGFR-3 coding sequence set forth in SEQ ID NO: 1. In one variation, anoligonucleotide probe of the invention is purified and isolated. Inanother variation, the oligonucleotide probe is labeled, e.g., with aradioisotope, chromophore, or fluorophore. In yet another variation, theprobe is covalently attached to a solid support [See generally Ausubelet al. And Sambrook et al., supra]

In preferred embodiments, the invention comprises an oligonucleotideprobe useful for detecting one or more of several mutations that havebeen characterized herein in affected individuals, including:

(1) a missense mutation at nucleotide 3360 of SEQ ID NO: 1, causing aproline to leucine change at residue 1114 in SEQ ID NO: 2;

(2) a missense mutation at nucleotide 2588 of SEQ ID NO: 1, causing aglycine to arginine change at residue 857 in SEQ ID NO: 2;

(3) a missense mutation at nucleotide 3141 of SEQ ID NO: 1, causing anarginine to proline change at residue 1041 in SEQ ID NO: 2;

(4) a missense mutation at nucleotide 3150 in SEQ ID NO: 1, causing aleucine to proline change at residue 1044 in SEQ ID NO: 2; and

(5) a missense mutation at nucleotide 3164 of SEQ ID NO: 1, causing anaspartic acid to asparagine change at residue 1049 in SEQ ID NO: 2.

For example, the invention provides oligonucleotides comprising anywherefrom 6 to 50 nucleotides that have a sequence that is identical to, orexactly complementary to, a portion of the human VEGFR-3 coding sequenceset forth in SEQ ID NO: 1, except for a nucleotide substitutioncorresponding to nucleotide 3360 of SEQ ID NO: 1. Such oligonucleotidesmay be generically described by the formula X_(n)YZ_(m) or itscomplement; where n and m are integers from 0 to 49; where 5≦(n+m)≦49;where X_(n) is a stretch of n nucleotides identical to a first portionof SEQ ID NO: 1 and Z_(m) is a stretch of m nucleotides identical to asecond portion of SEQ ID NO: 1, wherein the first and second portionsare separated in SEQ ID NO. 1 by one nucleotide; and wherein Yrepresents a nucleotide other than the nucleotide that separates thefirst and second portions of SEQ ID NO: 1. For example, where X_(m)represents 0 to 49 nucleotides immediately upstream (5′) of nucleotide3360 of SEQ ID NO: 1 and Z_(m) represents 0 to 49 nucleotidesimmediately downstream (3′) of nucleotide 3360 of SEQ ID NO: 1, Yrepresents a nucleotide other than cytosine, since a cytosine nucleotideis found at position 3360 of SEQ ID NO: 1. In a preferred embodiment, Yis a thymine nucleotide. Similar examples are contemplated for the otherspecific mutations identified immediately above.

In a related embodiment, the invention provides a kit comprising atleast two such oligonucleotide probes. Preferably, the two or moreprobes are provided in separate containers, or attached to separatesolid supports, or attached separately to the same solid support, e.g.,on a DNA microchip,

In still another related embodiment, the invention provides an array ofoligonucleotide probes immobilized on a solid support, the array havingat least 4 probes, preferably at least 100 probes, and preferably up to100,000, 10,000, or 1000 probes, wherein each probe occupies a separateknown site in the array. In a preferred embodiment, the array includesprobe sets comprising two to four probes, wherein one probe is exactlyidentical or exactly complementary to a human VEGFR-3 coding sequence,and the other one to three members of the set are exactly identical tothe first member, but for at least one different nucleotide, whichdifferent nucleotide is located in the same position in each of the oneto three additional set members. In one preferred embodiment, the arraycomprises several such sets of probes, wherein the sets correspond todifferent segments of the human VEGFR-3 gene sequence. In a highlypreferred embodiment, the array comprises enough sets ofoligonucleotides of length N to correspond to every particular N-mersequence of the VEGFR-3 gene, where N is preferably 6 to 25 and morepreferably 9 to 20. Materials and methods for making such probes areknown in the art and are described, for example, in U.S. Pat. Nos.5,837,832, 5,202,231, 5,002,867, and 5,143,854.

Moreover, the discoveries which underlie the present invention identifya target for therapeutic intervention in cases of hereditary lymphedema.The causative mutations in the families that have been studied ingreatest detail are mutations that appear to result in VEGFR-3 signalingthat is reduced in heterozygous affected individuals, but not completelyeliminated. This data supports a therapeutic indication foradministration of agents, such as VEGFR-3 ligand polypeptides, that willinduce VEGFR-3 signaling in the lymphatic endothelia of affectedindividuals to effect improvement in the structure and function of thelymphatic vasculature of such individuals. In addition, therapeutic genetherapy, to replace defective VEGFR-3 alleles or increase production ofVEGFR-3 ligand polypeptides in vivo, is envisioned as an aspect of theinvention.

Thus, in yet another aspect, the invention provides a therapeutic orprophylactic method of treatment for lymphedema, comprising the step ofadministering to a mammalian subject in need of therapeutic orprophylactic treatment for lymphedema a composition comprising acompound effective to induce intracellular signaling of VEGFR-3 inlymphatic endothelial cells that express said receptor. In a preferredembodiment, the compound comprises a polypeptide ligand for VEGFR-3, ora polynucleotide encoding such a ligand, wherein the polynucleotide isadministered in a form that results in transcription and translation ofthe polynucleotide in the mammalian subject to produce the ligand invivo. In another preferred embodiment, the compound comprises any smallmolecule that is capable of binding to the VEGFR-3 receptorextracellular or intracellular domain and inducing intracellularsignaling.

For example, the invention provides a therapeutic or prophylactic methodof treatment for lymphedema, comprising the step of administering to amammalian subject in need of therapeutic or prophylactic treatment forlymphedema a composition comprising a polynucleotide, the polynucleotidecomprising a nucleotide sequence that encodes a vascular endothelialgrowth factor C (VEGF-C) polypeptide. In a preferred embodiment, thesubject is a human subject.

While it is contemplated that the VEGF-C polynucleotide could beadministered purely as a prophylactic treatment to prevent lymphedema insubjects at risk for developing lymphedema, it is contemplated in apreferred embodiment that the polynucleotide be administered to subjectsafflicted with lymphedema, for the purpose of ameliorating its symptoms(e.g., swelling due to the accumulation of lymph). The polynucleotide isincluded in the composition in an amount and in a form effective topromote expression of a VEGF-C polypeptide in or near the lymphaticendothelia of the mammalian subject, to stimulate VEGFR-3 signaling inthe lymphatic endothelia of the subject.

In a preferred embodiment, the mammalian subject is a human subject.Practice of methods of the invention in other mammalian subjects,especially mammals that are conventionally used as models fordemonstrating therapeutic efficacy in humans (e.g., primate, porcine,canine, equine, murine, or rabbit animals), also is contemplated.Several potential animal models for hereditary lymphedema have beendescribed in the literature. [See, e.g., Lyon et al., Mouse News Lett.71: 26 (1984), Mouse News Lett. 74: 96 (1986), and Genetic variants andstrains of the laboratory mouse, 2nd ed., New York: Oxford UniversityPress (1989), p. 70 (Chylous ascites mouse), Dumont et al., Science,282: 946-949 (1998) (heterozygous VEGFR-3 knockout mouse), Patterson etal., “Hereditary Lymphedema,” Comparative Pathology Bulletin, 3: 2(1971) (canine hereditary lymphedema model); van der Putte, “CongenitalHereditary Lymphedema in the Pig,” Lympho. 11: 1-9 (1978), andCampbell-Beggs et al., “Chyloabdomen in a neonatal foal,” VeterinaryRecord, 137: 96-98 (1995).] Those models which are determined to haveanalogous mutations to the VEGFR-3 gene, such as the Chylous ascetei(Chy) mouse, are preferred. The present inventors have analyzed theVEGFR-3 genes of the Chy mouse and determined that affected mice containa missense mutation that results in a phenylalanine (rather than anisoleucine) in the VEGFR-3 sequence at a position corresponding to theisoleucine at position 1053 of SEQ ID NO: 2. This mutation maps to thecatalytic pocket region of the tyrosine kinase domain of the VEGFR-3protein, and may represent a viable model for identical mutations inhuman (if discovered) or other mutations in humans that similarly affectthe tyrosine kinase catalytic domain. The Chy mouse has peripheralswelling (oedema) after birth and chyle ascites. In another embodiment,“knock in” homologous recombination genetic engineering strategies areused to create an animal model (e.g., a mouse model) having a VEGFR-3allelic variation analogous to the human variations described herein.[See, e.g., Partanen et al., Genes & Development, 12: 2332-2344 (1998)(gene targeting to introduce mutations into a receptor protein (FGFR-1)in mice).] Such mice can also be bread to the heterozygous VEGFR-3knockout mice or Chy mice described above to further modify thephenotypic severity of the lymphedema disease.

For the practice of methods of the invention, the term “VEGF-Cpolypeptide” is intended to include any polypeptide that has a VEGF-C orVEGF-C analog amino acid sequence (as defined elsewhere herein ingreater detail) and that is able to bind the VEGFR-3 extracellulardomain and stimulate VEGFR-3 signaling in vivo. The term “VEGF-Cpolynucleotide” is intended to include any polynucleotide (e.g., DNA orRNA, single- or double-stranded) comprising a nucleotide sequence thatencodes a VEGF-C polypeptide. Due to the well-known degeneracy of thegenetic code, multiple VEGF-C polynucleotide sequences exist that encodeany selected VEGF-C polypeptide. Preferred VEGF-C polynucleotides,polypeptides, and VEGF-C variants and analogs for use in this inventionare disclosed in International Patent Application No. PCT/US98/01973,published as WO 98/33917, incorporated herein by reference in itsentirety.

For treatment of humans, VEGF-C polypeptides with an amino acid sequenceof a human VEGF-C are highly preferred, and polynucleotides comprising anucleotide sequence of a human VEGF-C cDNA are highly preferred. By“human VEGF-C” is meant a polypeptide corresponding to a naturallyoccurring protein (prepro-protein, partially-processed protein, orfully-processed mature protein) encoded by any allele of the humanVEGF-C gene, or a polypeptide comprising a biologically active fragmentof a naturally-occurring mature protein. By way of example, a humanVEGF-C comprises a continuous portion of the amino acid sequence setforth in SEQ ID NO: 4 sufficient to permit the polypeptide to bind andstimulate VEGFR-3 phosphorylation in cells that express such receptors.A polypeptide comprising amino acids 131-211 of SEQ ID NO: 4 isspecifically contemplated. For example, polypeptides having an aminoacid sequence comprising a continuous portion of SEQ ID NO: 4, thecontinuous portion having, as its amino terminus, an amino acid selectedfrom the group consisting of positions 30-131 of SEQ ID NO: 4, andhaving, as its carboxyl terminus, an amino acid selected from the groupconsisting of positions 211-419 of SEQ ID NO: 4 are contemplated. Anamino terminus selected from the group consisting of positions 102-131of SEQ ID NO: 4 is preferred, and an amino terminus selected from thegroup consisting of positions 103-113 of SEQ ID NO: 4 is highlypreferred. Likewise, a carboxyl terminus selected from the groupconsisting of positions 211-227 of SEQ ID NO: 4 is preferred. As statedabove, the term “human VEGF-C” also is intended to encompasspolypeptides encoded by allelic variants of the human VEGF-Ccharacterized by the sequences set forth in SEQ ID NOs: 3 & 4.

Moreover, since the therapeutic VEGF-C is to be administered asrecombinant VEGF-C or indirectly via somatic gene therapy, it is withinthe skill in the art to make and use analogs of human VEGF-C (andpolynucleotides that encode such analogs) wherein one or more aminoacids have been added, deleted, or replaced with other amino acids,especially with conservative replacements, and wherein theVEGFR-3-stimulatory biological activity has been retained. Analogs thatretain VEGFR-3-stimulatory VEGF-C biological activity are contemplatedas VEGF-C polypeptides for use in the present invention. In a preferredembodiment, analogs having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 such modifications andthat retain VEGFR-3-stimulatory VEGF-C biological activity arecontemplated as VEGF-C polypeptides for use in the present invention.Analogs having a deletion of or substitution for the cysteine residue atposition 156 of SEQ ID NO: 4 and that retain VEGFR-3 stimulatoryactivity but have reduced activity toward the receptor VEGFR-2, which isexpressed in blood vessels, are specifically contemplated. See WO98/33917. Polynucleotides encoding such analogs are generated usingconventional PCR site-directed mutagenesis, and chemical synthesistechniques.

Also contemplated as VEGF-C polypeptides are non-human mammalian oravian VEGF-C polypeptides and polynucleotides. By “mammalian VEGF-C” ismeant a polypeptide corresponding to a naturally occurring protein(prepro-protein, partially-processed protein, or fully-processed matureprotein) encoded by any allele of a VEGF-C gene of any mammal, or apolypeptide comprising a biologically active fragment of a matureprotein. The term “mammalian VEGF-C polypeptide” is intended to includeanalogs of mammalian VEGF-C's that possess the in vivoVEGFR-3-stimulatory effects of the mammalian VEGF-C.

Irrespective of which encoded VEGF-C polypeptide is chosen, any VEGF-Cpolynucleotide gene therapy pharmaceutical encoding it preferablycomprises a nucleotide sequence encoding a secretory signal peptidefused in-frame with the VEGF-C polypeptide sequence. The secretorysignal peptide directs secretion of the VEGF-C polypeptide by the cellsthat express the polynucleotide, and is cleaved by the cell from thesecreted VEGF-C polypeptide. For example, the VEGF-C polynucleotidecould encode the complete prepro-VEGF-C sequence set forth in SEQ ID NO:4, or could encode the VEGF-C signal peptide fused in-frame to asequence encoding a fully-processed VEGF-C (e.g., amino acids 103-227 ofSEQ ID NO: 4) or VEGF-C analog. Moreover, there is no requirement thatthe signal peptide be derived from VEGF-C. The signal peptide sequencecan be that of another secreted protein, or can be a completelysynthetic signal sequence effective to direct secretion in cells of themammalian subject.

In one embodiment, the VEGF-C polynucleotide of the invention comprisesa nucleotide sequence that will hybridize to a polynucleotide that iscomplementary to the human VEGF-C cDNA sequence specified in SEQ ID NO:3 under the following exemplary stringent hybridization conditions:hybridization at 42° C. in 50% formamide, 5×SSC, 20 mM Na·PO₄, pH 6.8;and washing in 1×SSC at 55° C. for 30 minutes: and wherein thenucleotide sequence encodes a polypeptide that binds and stimulateshuman VEGFR-3. It is understood that variation in these exemplaryconditions occur based on the length and GC nucleotide content of thesequences to be hybridized. Formulas standard in the art are appropriatefor determining appropriate hybridization conditions. [See Sambrook etal., Molecular Cloning: A Laboratory Manual (Second ed., Cold SpringHarbor, N.Y.: Cold Spring Harbor Laboratory Press, 1989) §§ 9.47-9.51.]

In preferred embodiments, the VEGF-C polynucleotide further comprisesadditional sequences to facilitate the VEGF-C gene therapy. In oneembodiment, a “naked” VEGF-C transgene (i.e., a transgene without aviral, liposomal, or other vector to facilitate transfection) isemployed for gene therapy. In this embodiment, the VEGF-C polynucleotidepreferably comprises a suitable promoter and/or enhancer sequence (e.g,cytomegalovirus promoter/enhancer [Lehner el al., J. Clin. Microbiol.,29:2494-2502 (1991); Boshart el al., Cell, 41-521-530 (1985)]; Roussarcoma virus promoter [Davis et al., Hum. Gene Ther., 4:151 (1993)];Tie promoter [Korhonen et al., Blood, 86(5) 1828-1835 (1995)]; or simianvirus 40 promoter) for expression in the target mammalian cells, thepromoter being operatively linked upstream (i.e., 5′) of the VEGF-Ccoding sequence. The VEGF-C polynucleotide also preferably furtherincludes a suitable polyadenylation sequence (e.g., the SV40 or humangrowth hormone gene polyadenylation sequence) operably linked downstream(i.e., 3′) of the VEGF-C coding sequence. The polynucleotide may furtheroptionally comprise sequences whose only intended function is tofacilitate large-scale production of the vector, e.g., in bacteria, suchas a bacterial origin of replication and a sequence encoding aselectable marker. However, in a preferred embodiment, such extraneoussequences are at least partially cleaved off prior to administration tohumans according to methods of the invention. One can manufacture andadminister such polynucleotides to achieve successful gene therapy usingprocedures that have been described in the literature for othertransgenes. See, e.g., Isner et al., Circulation, 91: 2687-2692 (1995);and Isner et al., Human Gene Therapy, 7: 989-1011 (1996); incorporatedherein by reference in the entirety.

Any suitable vector may be used to introduce the VEGF-C transgene intothe host. Exemplary vectors that have been described in the literatureinclude replication-deficient retroviral vectors, including but notlimited to lentivirus vectors [Kim et al., J. Virol, 72(1): 811-816(1998); Kingsman & Johnson, Scrip Magazine, October, 1998, pp. 43-46.]:adeno-associated viral vectors [Gnatenko et al., J. Investig. Med., 45:87-98 (1997)]; adenoviral vectors [See, e.g., U.S. Pat. No. 5,792,453;Quantin et al., Proc. Natl. Acad. Sci. USA, 89: 2581-2584 (1992);Stratford-Perricadet et al., J. Clin. Invest., 90: 626-630 (1992); andRosenfeld et al., Cell, 68: 143-155 (1992)]; Lipofectin-mediated genetransfer (BRL); liposomal vectors [See, e.g., U.S. Pat. No. 5,631,237(Liposomes comprising Sendai virus proteins)]; and combinations thereof.All of the foregoing documents are incorporated herein by reference inthe entirety. Replication-deficient adenoviral vectors constitute apreferred embodiment.

In embodiments employing a viral vector, preferred polynucleotides stillinclude a suitable promoter and polyadenylation sequence as describedabove. Moreover, it will be readily apparent that, in these embodiments,the polynucleotide further includes vector polynucleotide sequences(e.g., adenoviral polynucleotide sequences) operably connected to thesequence encoding a VEGF-C polypeptide.

Thus, in one embodiment the composition to be administered comprises avector, wherein the vector comprises the VEGF-C polynucleotide. In apreferred embodiment, the vector is an adenovirus vector. In a highlypreferred embodiment, the adenovirus vector is replication-deficient,i.e., it cannot replicate in the mammalian subject due to deletion ofessential viral-replication sequences from the adenoviral genome. Forexample, the inventors contemplate a method wherein the vector comprisesa replication-deficient adenovirus, the adenovirus comprising the VEGF-Cpolynucleotide operably connected to a promoter and flanked on eitherend by adenoviral polynucleotide sequences.

The composition to be administered according to methods of the inventionpreferably comprises (in addition to the polynucleotide or vector) apharmaceutically-acceptable carrier solution such as water, saline,phosphate-buffered saline, glucose, or other carriers conventionallyused to deliver therapeutics intravascularly. Multi-gene therapy is alsocontemplated, in which case the composition optionally comprises boththe VEGF-C polynucleotide/vector and another polynucleotide/vector. Asdescribed in greater detail below, a VEGF-D transgene is a preferredcandidate for co-administration with the VFGF-C transgene.

The “administering” that is performed according to the present methodmay be performed using any medically-accepted means for introducing atherapeutic directly or indirectly into a mammalian subject to reach thelymph or the lymphatic system, including but not limited to injections;oral ingestion, intranasal or topical administration; and the like. In apreferred embodiment, administration of the composition comprising theVEGF-C polynucleotide is performed intravascularly, such as byintravenous or intra-arterial injection, or by subcutaneous injection orlocal depot administration. In a highly preferred embodiment, thecomposition is administered locally, e.g., to the site of swelling.

In still another variation, endothelial cells or endothelial progenitorcells are transfected ex vivo with a wild type VEGFR-3 transgene, andthe transfected cells are administered to the mammalian subject.

In another aspect, the invention provides a therapeutic or prophylacticmethod of treating for lymphedema, comprising the step of administeringto a mammalian subject in need of treatment for lymphedema a compositioncomprising a VEGF-C polypeptide, in an amount effective to treat orprevent swelling associated with lymphedema. Administration via one ormore intravenous or subcutaneous injections is contemplated.Co-administration of VEGF-C polynucleotides and VEGF-C polypeptides isalso contemplated.

In yet another embodiment, the invention provides the use of a VEGF-Cpolynucleotide or VEGF-C polypeptide for the manufacture of a medicamentfor the treatment or prevention of lymphedema.

In still another embodiment, the invention provides a therapeutic orprophylactic method of treatment for lymphedema, comprising the step ofadministering to a mammalian subject in need of therapeutic orprophylactic treatment of lymphedema a composition comprising apolynucleotide, the polynucleotide comprising a nucleotide sequence thatencodes a vascular endothelial growth factor D (VEGF-D) polypeptide.Such methods are practiced essentially as described herein with respectto VEGF-C-encoding polynucleotides, except that polynucleotides encodingVEGF-D are employed. A detailed description of the human VEGF-D gene andprotein are provided in Achen, et al., Proc. Nat'l Acad. Sci. USA.,95(2): 548-553 (1998); International Patent Publication No. WO 98/07832,published 26 Feb. 1998, and in Genbank Accession No. AJ000185, allincorporated herein by reference. A cDNA and deduced amino acid sequencefor prepro-VEGF-D is set forth herein in SEQ ID NOs: 5 and 6. Of course,due to the well-known degeneracy of the genetic code, multiple VEGF-Dencoding polynucleotide sequence exist, any of which may be employedaccording to the methods taught herein.

As described herein in detail with respect to VEGF-C, the use ofpolynucleotides that encode VEGF-D fragments, VEGF-D analogs, VEGF-Dallelic and interspecies variants, and the like which possess in vivostimulatory effects of human VEGF-D are all contemplated as beingencompassed by the present invention.

In yet another embodiment, the invention provides a therapeutic orprophylactic method of treatment for lymphedema, comprising the step ofadministering to a mammalian subject in need of treatment for lymphedemaa composition comprising a VEGF-D polypeptide, in an amount effective totreat or prevent swelling associated with lymphedema. Administration viaone or more intravenous or subcutaneous injections is contemplated.

The VEGFR-3 allelic variant polynucleotides and polypeptides describedherein that were discovered and characterized by the present inventorsare themselves considered aspects of the invention. Such polynucleotidesand polypeptides are useful, for example, in screening assays (e.g.,cell-based assays or assays involving transgenic mice that express thepolynucleotide in lieu of a native VEGF-3 allele) to study thebiological activities of VEGFR-3 variant alleles and identify compoundsthat are capable of modulating that activity, e.g., to identifytherapeutic candidates for treatment of lymphedema. Such screeningassays are also considered aspects of the invention.

The polypeptides of the invention are intended to include completeVEGFR-3 polypeptides with signal peptide (e.g., approximately residues 1to 20 of SEQ ID NO: 2), mature VEGFR-3 polypeptides lacking any signalpeptide, and recombinant variants wherein a foreign or synthetic signalpeptide has been fused to the mature VEGFR-3 polypeptide.Polynucleotides of the invention include all polynucleotides that encodeall such polypeptides. It will be understood that for essentially anypolypeptide, many polynucleotides can be constructed that encode thepolypeptide by virtue of the well known degeneracy of the genetic code.All such polynucleotides are intended as aspects of the invention.

Thus, in yet another aspect, the invention provides a purifiedpolynucleotide comprising a nucleotide sequence encoding a human VEGFR-3protein variant, wherein said polynucleotide is capable of hybridizingto the complement of SEQ ID NO: 1 under stringent hybridizationconditions, and wherein the encoded VEGFR-3 protein variant has an aminoacid sequence that differs at position 1114, 857, 1041, 1044 or 1049from the amino acid sequence set forth in SEQ ID NO: 1. Exemplaryconditions are as follows: hybridization at 42° C. in 50% formamide,5×SSC, 20 mM Na·PO4, pH 6.8; and washing in 0.2×SSC at 55° C. It isunderstood by those of skill in the art that variation in theseconditions occurs based on the length and GC nucleotide content of thesequences to be hybridized. Formulas standard in the art are appropriatefor determining appropriate hybridization conditions. [See Sambrook etal. (1989), supra, §§ 9.47-9.51.]

In a related embodiment, the invention provides a purifiedpolynucleotide comprising a nucleotide sequence encoding a VEGFR-3protein of a human that is affected with heritable lymphedema or otherlymphatic disorder; wherein the polynucleotide is capable of hybridizingto the complement of SEQ ID NO: 1 under stringent hybridizationconditions, and wherein the encoded polynucleotide has an amino acidsequence that differs from SEQ ID NO: 1 at at least one codon. It willbe understood that conventional recombinant techniques can be used toisolate such polynucleotides from individuals affected with heritablelymphedema or their relatives. The wildtype VEGFR-3 cDNA sequence setforth in SEQ ID NO: 1 (or its complement, or fragments thereof) is usedas a probe to identify and isolate VEGFR-3 sequences from nucleic acidderived from the individuals. Alternatively, PCR amplification primersbased on the wildtype VEGFR-3 sequence are generated and used to amplifyeither VEGFR-3 genomic DNA or VEGFR-3 mRNA from the human subject. Theresultant amplified genomic DNA or cDNA is sequenced to determine thevariations that characterize the VEGFR-3 lymphedema allele of theindividual. Preferred VEGFR-3 lymphedema alleles include, but are notlimited to the P1114L, G857R, R1041P, L1044P and D1049N allelesdescribed in detail herein.

In addition, the invention provides vectors that comprise thepolynucleotides of the invention. Such vectors are useful for amplifyingand expressing the VEGFR-3 proteins encoded by the polynucleotides, andfor creating recombinant host cells and/or transgenic animals thatexpress the polynucleotides. The invention further provides a host celltransformed or transfected with polynucleotides (including vectors) ofthe invention. In a preferred embodiment, the host cell expresses theencoded VEGFR-3 protein on its surface. Such host cells are useful incell-based screening assays for identifying modulators that stimulate orinhibit signaling of the encoded VEGFR-3. Modulators that stimulateVEGFR-3 signaling have utility as therapeutics to treat lymphedemas,whereas modulators that are inhibitory have utility for treatinghyperplastic lymphatic conditions mediated by the allelic variantVEGFR-3. In a preferred embodiment, host cells of the invention areco-transfected with both a wildtype and an allelic variant VEGFR-3polynucleotide, such that the cells express both receptor types on theirsurface. Such host cells are preferred for simulating a heterozygousVEGFR-3 genotype of many individuals affected with lymphedema.

In yet another aspect, the invention provides a transgenic mammal, e.g.,mouse, characterized by a non-native VEGFR-3 allele that has beenintroduced into the mouse, and the transgenic progeny thereof. Preferredallelic variants include allelic variants that correlate with hereditarylymphedema in human subjects, such as an allelic variant wherein aP1114L, G857R, R1041P, L1044P or D1049N missense mutation has beenintroduced into the murine VEGFR-3 gene, or wherein the human P1114L,G857R, R1041P, L1044P or D1049N allelic variant has been substituted fora murine VEGFR-3 allele. Such mice are produced using standard methods.[See, e.g., Hogan et al. (eds.), Manipulating the Mouse Embryo, ColdSpring Harbor, N.Y.: Cold Spring Harbor Laboratory (1986).] Theintroduction of the human-like mutations into non-human sequences isreadily achieved with standard techniques, such as site-directedmutagenesis. The determination of which residues in a non-human sequenceto alter to mimic the foregoing human mutations is routine since theforegoing mutations all occur in regions of the VEGFR-3 sequence thatcontain residues that are highly conserved between species. See FIGS.3A-3B.

In yet another aspect, the invention provides assays for identifyingmodulators of VEGFR-3 signaling, particularly modulators of thesignaling of allelic variants of VEGFR-3 that correlate with lymphaticdisorders such as heritable lymphedema. For example, the inventionprovides a method for identifying a modulator of intracellular VEGFR-3signaling, comprising the steps of contacting a cell expressing at leastone mutant mammalian VEGFR-3 polypeptide in the presence and in theabsence of a putative modulator compound; b) detecting VEGFR-3 signalingin the cell, and c) identifying a putative modulator compound in view ofdecreased or increased signaling in the presence of the putativemodulator, as compared to signaling in the absence of the putativemodulator.

By “mutant mammalian VEGFR-3 polypeptide” is meant a VEGFR-3 polypeptidethat varies from a wildtype mammalian VEGFR-3 polypeptide (e.g., byvirtue of one or more amino acid additions, deletions, orsubstitutions), wherein the variation is reflective of a naturallyoccurring variation that has been correlated with a lymphatic disorder,such as lymphedema. By way of example, the previously describedsubstitution variations of human VEGFR-3, such as P1114L, have beencorrelated with heritable lymphedema. Any of the human allelic variantsdescribed above, or analogous human allelic variants having a differentsubstitution at the indicated amino acid positions, or a non-humanVEGFR-3 into which a mutation at the position corresponding to any ofthe described positions has been introduced are all examples of mutantmammalian VEGFR-3 polypeptides.

The detecting step can entail the detection of any parameter indicativeof VEGFR-3 signaling. For example, the detecting step can entail ameasurement of VEGFR-3 autophosphorylation, or a measurement ofVEGFR-3-mediated cell growth, or a measurement of any step in theVEGFR-3 signaling cascade between VEGFR-3 autophosphorylation and cellgrowth.

In a preferred embodiment, the method is practiced with a cell thatexpresses the mutant mammalian VEGFR-3 polypeptide and a wildtypemammalian VEGFR-3 polypeptide. Such cells are thought to better mimicthe conditions in heterozygous individuals suffering from aVEGFR-3-mediated lymphatic disorder. In a highly preferred embodiment,the mutant and wildtype VEGFR-3 polypeptides are human. In the preferredembodiments, the mutant VEGFR-3 polypeptide comprises a leucine aminoacid at the position corresponding to position 1114 of SEQ ID NO: 2; anarginine at the position corresponding to position 857 of SEQ ID NO: 2;a proline amino acid at the position corresponding to position 1041 ofSEQ ID NO: 2; a proline amino acid at the position corresponding toposition 1044 of SEQ ID NO: 2; or an asparagine at the positioncorresponding to position 1049 of SEQ 11 NO: 2.

Additional features and variations of the invention will be apparent tothose skilled in the art from the entirety of this application,including the drawing and detailed description, and all such featuresare intended as aspects of the invention. Likewise, features of theinvention described herein can be re-combined into additionalembodiments that are also intended as aspects of the invention,irrespective of whether the combination of features is specificallymentioned above as an aspect or embodiment of the invention. Also, onlysuch limitations which are described herein as critical to the inventionshould be viewed as such; variations of the invention lackinglimitations which have not been described herein as critical areintended as aspects of the invention.

In addition to the foregoing, the invention includes, as an additionalaspect, all embodiments of the invention narrower in scope in any waythan the variations specifically mentioned above. Although theapplicant(s) invented the full scope of the claims appended hereto, theclaims appended hereto are not intended to encompass within their scopethe prior art work of others. Therefore, in the event that statutoryprior art within the scope of a claim is brought to the attention of theapplicants by a Patent Office or other entity or individual, theapplicant(s) reserve the right to exercise amendment rights underapplicable patent laws to redefine the subject matter of such a claim tospecifically exclude such statutory prior art or obvious variations ofstatutory prior art from the scope of such a claim. Variations of theinvention defined by such amended claims also are intended as aspects ofthe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F depict pedigrees of six hereditary lymphedema families(Families 101, 106, 111, 135, 105, and 127, respectively) informativefor linkage. Filled symbols represent individuals with clinicallydocumented lymphedema. Crossed symbols represent individuals with anambiguous phenotype. An ambiguous phenotype is defined as self-reportedswelling of the limbs with no known cause, without a clinical diagnosisof lymphedema. Individuals of ambiguous phenotype were coded as diseasestatus unknown for the linkage analysis. The proband in each family isindicated by an arrow.

FIG. 2 is a graph summarizing VITESSE analysis of lymphedema familieswith markers localized to chromosome 5q34-q35. In the graph, filledcircles represent analyses for Families 101, 105, 106, and 111, openboxes represent analyses for Families 101, 106, and 111, open circlesrepresent the VEGFR-3 gene; and open triangles represent Family 135. Theone LOD confidence interval lies completely within the interval flankedby markers D5S1353 and D5S408 and overlaps the most likely location ofFlt4 (VEGFR-3). Linkage is excluded for the entire region for family135.

FIG. 3A-3B depict an alignment of portions of the human (top line, SEQID NO: 2) and murine (bottom line, Genbank Acc. No. P35917, SEQ ID NO:19) VEGFR-3 amino acid sequences to demonstrate similarity. Identicalresidues are marked with a line, and highly conserved and less conserveddifferences are marked with two dots or a single dot, respectively. Thelocation of various mutations that have been observed to correlate witha heritable lymphedema phenotype are indicated immediately beneath thealigned sequences.

DETAILED DESCRIPTION OF THE INVENTION

Certain therapeutic aspects of the present invention involve theadministration of Vascular Endothelial Growth Factor C or Dpolynucleotides and polypeptides. The growth factor VEGF-C, as well asnative human, non-human mammalian, and avian polynucleotide sequencesencoding VEGF-C, and VEGF-C variants and analogs, have been described indetail in International Patent Application Number PCT/US98/01973, filed02 Feb. 1998 and published on 06 Aug. 1998 as International PublicationNumber WO 98/33917; in Joukov et al., J. Biol. Chem., 273(12): 6599-6602(1998); and in Joukov et al., EMBO J., 16(13): 3898-3911 (1997), all ofwhich are incorporated herein by reference in the entirety. As explainedtherein in detail, human VEGF-C is initially produced in human cells asa prepro-VEGF-C polypeptide of 419 amino acids. A cDNA and deduced aminoacid sequence for human prepro-VEGF-C are set forth in SEQ ID NOs: 3 and4, respectively, and a cDNA encoding human VEGF-C has been depositedwith the American Type Culture Collection (ATCC), 10801 UniversityBlvd., Manassas, Va. 20110-2209 (USA), pursuant to the provisions of theBudapest Treaty (Deposit date of 24 Jul. 1995 and ATCC Accession Number97231). VEGF-C sequences from other species have also been reported. SeeGenbank Accession Nos. MMU73620 (Mus musculus); and CCY15837 (Coturnixcoturnix) for example, incorporated herein by reference.

The prepro-VEGF-C polypeptide is processed in multiple stages to producea mature and most active VEGF-C polypeptide of about 21-23 kD (asassessed by SDS-PAGE under reducing conditions). Such processingincludes cleavage of a signal peptide (SEQ ID NO: 4, residues 1-31),cleavage of a carboxyl-terminal peptide (corresponding approximately toamino acids 228-419 of SEQ ID NO: 4 and having a pattern of spacedcysteine residues reminiscent of a Balbiani ring 3 protein (BR3P)sequence [Dignam et al., Gene, 88:133-40 (1990); Paulsson et al., J.Mol. Biol., 211:331-49 (1990)] to produce a partially-processed form ofabout 29 kD, and cleavage (apparently extracellularly) of anamino-terminal peptide (corresponding approximately to amino acids32-103 of SEQ ID NO: 4) to produce a fully-processed mature form ofabout 21-23 kD. Experimental evidence demonstrates thatpartially-processed forms of VEGF-C (e.g., the 29 kD form) are able tobind the VEGFR-3 receptor, whereas high affinity binding to VEGFR-2occurs only with the fully processed forms of VEGF-C.

Moreover, it has been demonstrated that amino acids 103-227 of SEQ IDNO: 4 are not all critical for maintaining VEGF-C functions. Apolypeptide consisting of amino acids 113-213 (and lacking residues103-112 and 214-227) of SEQ ID NO: 2 retains the ability to bind andstimulate VEGFR-3, and it is expected that a polypeptide spanning fromabout residue 131 to about residue 211 will retain VEGF-C biologicalactivity. The cysteine residue at position 156 has been shown to beimportant for VEGFR-2 binding ability. However, VEGF-C ΔC₁₅₆polypeptides (i.e., analogs that lack this cysteine due to deletion orsubstitution) remain potent activators of VEGFR-3, and are thereforeconsidered to be among the preferred candidates for treatment oflymphedema. (It has been shown that a VEGF-C C156S serine substitutionanalog promotes lymphatic growth when over-expressed in the skin oftransgenic mice behind the K14 promote, in a manner analogous to whatwas described in Jeltsch et al., Science, 276:1423 (1997), incorporatedherein by reference.) The cysteine at position 165 of SEQ ID NO: 4 isessential for binding to either receptor, whereas analogs lacking thecysteines at positions 83 or 137 compete with native VEGF-C for bindingwith both receptors and are able to stimulate both receptors.

An alignment of human VEGF-C with VEGF-C from other species (performedusing any generally accepted alignment algorithm) suggests additionalresidues wherein modifications can be introduced (e.g., insertions,substitutions, and/or deletions) without destroying VEGF-C biologicalactivity. Any position at which aligned VEGF-C polypeptides of two ormore species have different amino acids, especially different aminoacids with side chains of different chemical character, is a likelyposition susceptible to modification without concomitant elimination offunction. An exemplary alignment of human, murine, and quail VEGF-C isset forth in FIG. 5 of PCT/US98/01973.

Apart from the foregoing considerations, it will be understood thatinnumerable conservative amino acid substitutions can be performed to awildtype VEGF-C sequence which are likely to result in a polypeptidethat retains VEGF-C biological activities, especially if the number ofsuch substitutions is small. By “conservative amino acid substitution”is meant substitution of an amino acid with an amino acid having a sidechain of a similar chemical character. Similar amino acids for makingconservative substitutions include those having an acidic side chain(glutamic acid, aspartic acid); a basic side chain (arginine, lysine,histidine); a polar amide side chain (glutamine, asparagine); ahydrophobic, aliphatic side chain (leucine, isoleucine, valine, alanine,glycine); an aromatic side chain (phenylalanine, tryptophan, tyrosine);a small side chain (glycine, alanine, serine, threonine, methionine); oran aliphatic hydroxyl side chain (serine, threonine). Addition ordeletion of one or a few internal amino acids without destroying VEGF-Cbiological activities also is contemplated.

Without intending to be limited to a particular theory, the mechanismbehind the efficacy of VEGF-C in treating or preventing lymphedema isbelieved to relate to the ability of VEGF-C to stimulate VEGFR-3signaling. Administration of VEGF-C in quantities exceeding thoseusually found in interstitial fluids is expected to stimulate VEGFR-3 inhuman subjects who, by virtue of a dominant negative heterozygousmutation, have insufficient VEGFR-3 signaling.

The growth factor named Vascular Endothelial Growth Factor D (VEGF-D),as well as human sequences encoding VEGF-D, and VEGF-D variants andanalogs, have been described in detail in International PatentApplication Number PCT/US97/14696, filed 21 Aug. 1997 and published on26 Feb. 1998 as International Publication Number WO 98/07832; and inAchen, et al., Proc. Nat'l Acad. Sci. U.S.A., 95(2): 548-553 (1998),both incorporated herein by reference in the entirety. As explainedtherein in detail, human VEGF-D is initially produced in human cells asa prepro-VEGF-D polypeptide of 354 amino acids. A cDNA and deduced aminoacid sequence for human prepro-VEGF-D are set forth in SEQ ID Nos: 5 and6, respectively. VEGF-D sequences from other species also have beenreported. See Genbank Accession Nos. D89628 (Mus musculus); and AF014827(Rattus norvegicus), for example, incorporated herein by reference.

The prepro-VEGF-D polypeptide has a putative signal peptide of 21 aminoacids and is apparently proteolytically processed in a manner analogousto the processing of prepro-VEGF-C. A “recombinantly matured” VEGF-Dlacking residues 1-92 and 202-354 of SEQ ID NO: 6 retains the ability toactivate receptors VEGFR-2 and VEGFR-3, and appears to associate asnon-covalently linked dimers. Thus, preferred VEGF-D polynucleotidesinclude those polynucleotides that comprise a nucleotide sequenceencoding amino acids 93-201 of SEQ ID NO: 6.

The subject matter of the invention is further described anddemonstrated with reference to the following examples.

EXAMPLE 1

Demonstration that Hereditary Lymphedema is linked to the VEGFR-3 Locus

The following experiments, conducted to identify a gene or genescontributing to susceptibility to develop lymphedema, demonstrated thathereditary lymphedema correlates, in at least some families, to thechromosomal locus for the VEGFR-3 gene.

OVERVIEW

Families with inherited lymphedema were identified for the purpose ofconducting a linkage and positional candidate gene analysis. Thirteendistinct families from the United States and Canada were identifiedthrough referrals from lymphedema treatment centers, lymphedema supportgroups, and from internet correspondence (worldwide web site atwww.pitt.edu/˜genetics/lymph/). The study protocol was approved by theInstitutional Review Board of the University of Pittsburgh andparticipants gave written informed consent. All members of the familieswere of western European ancestry. Forty members of one family (“Family101”) were examined during a family reunion by a psychiatristexperienced in lymphedema treatment. Family members were consideredaffected with hereditary lymphedema if they exhibited asymmetry orobvious swelling of one or both legs. Members of the other 12 familieswere scored as affected if they had received a medical diagnosis oflymphedema, or if there were personal and family reports of extremityswelling or asymmetry. Medical records were obtained to verify statuswhenever possible. For the purpose of linkage analysis, individuals withvery mild or intermittent swelling, heavyset legs, obesity, or a historyof leg infections as the only symptom were considered to haveindeterminate disease status.

In the 13 families, 105 individuals were classified as affected, with amale:female ratio of 1:2.3. The age of onset of lymphedema symptomsranged from prenatal (diagnosed by ultrasound) to age 55. When affectedby normal matings were analyzed, 76 of 191 children were affected,yielding a penetrance of 80%. First degree relatives of affectedindividuals were considered at risk.

Biological samples were obtained from members of the thirteen familiesto conduct the genetic analyses. DNA was isolated from theEDTA-anticoagulated whole blood by the method of Miller et al., NucleicAcids Res., 16: 1215 (1998), and from cytobrush specimens using thePuregene DNA isolation kit (Gentra Systems, Minneapolis, Minn.).Analysis of the markers used in the genome scan were performed bymethods recognized in the art. See Browman et al., Am. J. Hum. Genetic.,63:861-869 (1998); see also the NHLBI Mammalian Genotyping Serviceworld-wide web sites (www.marshmed.org/genetics/methods/pcr.htm; andwww.marshmed.org/genetics/methods/gel.htm).

Two-point linkage analysis was conducted using an autosomal dominantmodel predicting 80% penetrance in the heterozygous state, 99%penetrance in the homozygous state, and a 1% phenocopy rate. Thefrequency of the disease allele was set at 1/10,000. Microsatellitemarker allele frequencies were calculated by counting founder alleles,with the addition of counts of non-transmitted alleles. Multipointanalysis was carried out using distances obtained from the LocationDatabase (LDB-http://cedar.genetics.soton.ac.uk/public html). Multipointand 2-point analyses were facilitated using the VITESSE (v1.1) program.[O'Connell, J. R. and Weeks, D. E., (1995), Nature Genet., 11:402-408].

DETAILED DESCRIPTION OF METHODS AND RESULTS

The first family studied, Family 101, was a large, multi-generationalfamily demonstrating early onset lymphedema, (See FIG. 1). Fortyindividuals of this family were examined and DNA sampled. In addition,blood was obtained from another 11 members from mailing kits. Linkagesimulation was performed using SLINK [Weeks et al., Am. J. Hum. Genet.47:A204 (1990)] and linkage was analyzed using MSIM [Ott, J., Proc. Nat.Acad. Sci. USA, 86:4175-4178 (1989)] to estimate the potential power oftwo point linkage analysis in the family. Marker genotypes weresimulated for a marker with heterozygosity of 0.875 under a linked (θ=0)and unlinked (θ=0.5) model using the 51 available individuals. Thesimulation showed that the power to detect linkage was greater than 90%for a LOD score threshold of Z(θ) 2.0. The false positive rate was lessthan 5%.

Shortly thereafter, two additional families (designated Families 106 and111) segregating for autosomal dominant lymphedema were identified.These three families (FIGS. 1A-1C, Families 101, 106 and 111) weregenotyped for 366 autosomal markers by the NHLBI Mammalian GenotypingService (www.marshmed.org/genetics). Genotypes were checked forconsistency using Pedcheck [O'Connell, J. R. and Weeks, D. E., Am. J.Hum. Genet., 61:A288 (1997)]. Two point linkage analysis was performedusing VITESSE [O'Connell, J. R. and Weeks, D. E., Nature Genet.,11:402-408 (1995)]. The model for linkage assumed an autosomal dominantmodel of inheritance, a disease allele frequency of 0.0001 and apenetrance of 0.80.

The results from the genomic scan can be briefly summarized as follows.A summed LOD score of greater than 4.0 was observed from distalchromosome 5, markers D5S1456, D5S817 and D5S488. The markers on distalchromosome 5q were the only markers having Z>3.0, the criteriaestablished for statistical significance. LOD scores greater than 2.0(θ=0-0.15) were also detected for chromosome 12 (D12S391 Z=2.03, allfamilies), and chromosome 21 (D21S1440 Z=2.62, all families). Thelargest two-point LOD (Z=4.3; θ=0) was observed for marker D5S408,localized to chromosome 5q34-q35.

This initial chromosomal mapping was further refined by genotyping thethree affected families for eight additional markers localized to region5q34-q35. Six of these were informative for linkage (D5S653, D5S498,D5S408, D5S2006, D5S1353 and D5S1354). Linkage analysis of these markersusing VlTESSE yielded a 2-point LOD score of 6.1 at θ=0 for markerD5S1354 (Table 1) and a maximum multipoint LOD score of 8.8 at markerD5S1354 (FIG. 2). These findings supported the localization of a genewithin chromosome band 5q34-q35 that is a predisposing factor inhereditary lymphedema. TABLE 1 LOD scores for individual familiesestimated over the interval defined by markers D5S498 and D5S2006. Z(θ)0.0 Z(θ) 0.01 Z(θ) 0.05 Z(θ) 0.1 Z(θ) 0.2 Locus D5S498 Family 101 −3.18−2.33 −0.45 0.42 0.88 Family 106 1.08 1.07 1.05 0.99 0.81 Family 111−0.85 −0.77 −0.53 −0.34 −0.13 Family 105 1.22 1.20 1.11 0.98 0.72 Family135 −2.48 −1.85 −1.12 −0.75 −0.38 Locus D5S1353 Family 101 −2.99 −2.48−1.21 −0.63 −0.18 Family 106 0.28 0.29 0.35 0.38 0.38 Family 111 −1.06−1.02 −0.88 −0.72 −0.42 Family 105 0.72 0.71 0.65 0.56 0.39 Family 135−8.03 −4.18 −2.09 −1.13 −0.30 Locus D5S1354 Family 101 6.09 6.02 5.695.21 4.07 Family 106 1.42 1.40 1.32 1.20 0.96 Family 111 0.21 0.22 0.230.24 0.22 Family 105 0.43 0.42 0.40 0.36 0.28 Family 135 −6.88 −4.91−3.20 −2.16 −1.07 Locus D5S408 Family 101 2.80 2.74 2.50 2.20 1.56Family 106 0.66 0.68 0.73 0.76 0.71 Family 111 −1.70 −1.40 −0.80 −0.44−0.10 Family 105 0.42 0.41 0.38 0.35 0.27 Family 135 −5.22 −4.24 −2.58−1.67 −0.80 Locus D5S2006 Family 101 4.51 4.70 4.85 4.66 3.80 Family 1061.17 1.16 1.11 1.03 0.83 Family 111 −1.32 −1.18 −0.82 −0.56 −0.25 Family105 0.43 0.42 0.40 0.36 0.28 Family 135 −3.86 −3.20 −2.11 −1.45 −0.73

During the completion of the genome scan, an additional ten lymphedemafamilies were ascertained. Two of these families (Families 105 and 135,see FIGS. 1E and 1D), were potentially informative for linkage and weregenotyped for markers in the linked region. Examination of the two pointLOD scores for the five informative families for markers in the linkedregion (Table 1) shows that four of the families (101, 105, 106 and 111)are consistent with linkage to chromosome 5q while family 135 excludedlinkage across the entire region with LOD scores Z=<−2.0 for allmarkers. Multipoint linkage analysis of Families 101, 105, 106 and 111(FIG. 2) yielded a peak LOD score of Z=10 at marker D5S1354. Thesefindings support the existence of at least two loci which predispose tohereditary lymphedema.

The order of markers D5S1353, D5S1354 and D5S408 with respect to eachother was uncertain. Multipoint linkage analysis using alternativeorders for these markers gave similar results. Marker D5S498 is aframework marker and marker D5S408 is mapped 11.2 centimorgans distal toD5S498, based on the CHLC chromosome 5 sex averaged, recombinationminimized map, version 3 (www.chlc.org). The physical distance betweenD5S498 and D5S408 is estimated as 1.45 megabases based on the GeneticLocation Database (LDB) chromosome 5 summary map(cedar.genetics.soton.ac.uk/public_html/).

Database analysis identified sixteen genes within this region. Two ofthese genes have been identified as having roles in development (MSX2and VEGFR-3). MSX2 was considered an unlikely candidate gene forlymphedema because of its known involvement in craniofacial development[Jabs et al., Cell, 75: 443-450 (1993)]. VEGFR-3, the gene encoding areceptor for VEGF-C, was selected as a better candidate gene for initialfurther study for the following reasons.

(1) VEGFR-3 is expressed in developing lymphatic endothelium in themouse [Kukk et al., Development, 122: 3829-3837 (1996); and Kaipainen etal., Proc. Nat. Acad. Sci. USA, 92: 3566-3570 (1995)].

(2) expression of VEGFR-3 is induced in differentiating avianchorioallantoic membrane [Oh et al., Dev. Biol., 188:96-109 (1997)]; and

(3) overexpression of VEGF-C, a ligand of VEGFR-3, leads to heperplasiaof the lymphatic vessels in transgenic mice [Jeltsch et al., Science,276: 1423-1425 (1997)].

To explore the potential role of VEGFR-3 in lymphedema, probands fromthe thirteen lyomphedema families were screened for variation by directsequencing of portions of the VEGFR-3 gene. The sequencing strategy usedamplification primers generated based upon the VEGFR-3 cDNA sequence(SEQ ID NO: 1) and information on the genomic organization of therelated vascular endothelial growth factor receptor-2(VEGFR-2/KDR/flk-1) [Yin et al., Mammalian Genome, 9: 408-410 (1998)].Variable positions (single nucleotide polymorphisms), the uniquesequence primers used to amplify sequences flanking each variable site,and the method of detecting each variant are summarized in Table 2.TABLE 2 Location, amplification primer sequences, amplificationconditions, and detection methods for five intragenic single nucleotidepolymorphisms in the human VEGFR-3 gene Position Ann. Base Detection inVEGFR-3 gene Primer 1 sequence Primer 2 sequence temp. [MgCl₂] changeMethod Exon 12, amino tcaccatcgatccaagc agttctgcgtgagccgag 56° C. 1.0 mMC-T Sequencing acid 641 (SEQ ID NO: 7) (SEQ ID NO: 8) Exon 24, aminocaggacggggtgacttga gcccaggcctgtctactg 56° C. 1.0 mM C-T Sequencing acid1114 (SEQ ID NO: 9) (SEQ ID NO: 10) Exon 3, amino ccagctcctacgtgttcgggcaacagctggatgtca 56° C. 1.0 mM C-T Hhal acid 175 (SEQ ID NO: 11) (SEQID NO: 12) 65 bp 3′ ctgtgagggcgtgggagt gtcctttgagccactgga 54° C. 1.5 mMG-A Styl to Exon 6 (SEQ ID NO: 13) (SEQ ID NO: 14) 55 bp 3′cacacgtcatcgacaccggtg ggcaacagctggatgtca 56° C. 1.5 mM C-T Apal to Exon2 (SEQ ID NO: 15) (SEQ ID NO: 16)All amplifications were done for 35 cycles with denaturation at 94° for30 seconds, annealing as above for 30 seconds, and extension at 72° for30 seconds.Amplification and sequencing primers were synthesized by the DNASynthesis Facility, University of Pittsburgh. Amplification primers weretagged at the 5′ end with the forward or reverse M13 universal sequenceto facilitate direct sequencing. Amplifiers were subjected to cyclesequencing using the dRhodamine terminator ready reaction kit or the DyePrimer ready reaction kit for -M13 and M13 Rev primers (Perkin Elmer)and analyzed on the Prism ABI 377 fluorescent sequencer. Sequences werealigned for further analysis using SEQUENCER 3.0 (Gene Codes).

Genomic sequence from approximately 50% of the VEGFR-3 gene wasdetermined in this manner, and five single nucleotide variants wereobserved. Two of the variants occurred in introns, and a third was asilent substitution in predicted exon 3. These intragenic polymorphismswere used to map the VEGFR-3 gene. As shown in FIG. 2, VEGFR-3 mapswithin the region of chromosome 5q linked to the lymphedema phenotype,consistent with it being selected as a candidate gene. In two families,(Family 127 pedigree not shown, and Family 135), a C-T transition wasidentified at nucleotide position 1940 of the VEGFR-3 cDNA (SEQ ID NO:1). This nucleotide substitution is predicted to lead to anon-conservative substitution of serine (codon TCC) for proline (codonCCC) at residue 641 (putative exon 12, within the sixthimmunoglobulin-like region of the receptor's extracellular domain) ofthe amino acid sequence of the receptor (SEQ ID NO: 2). However, thissequence change was observed in 2 of 120 randomly selected individualsfrom the general population (240 alleles). Also, in one of the twofamilies in which this variant was initially detected, family 135,linkage between lymphedema and chromosome 5q markers was excludes (Table1 and FIG. 2). In probands from the other ten families, wild typesequence was observed at nucleotide position 1940. Collectively, theseresults suggest that this P641S variant is not causative.

In one nuclear family (Family 127, pedigree shown in FIG. 1F) a C-Ttransition was observed at nucleotide position 3360 (SEQ ID NO: 1) ofthe VEGFR-3 cDNA. This nucleotide substitution is predicted to lead to anon-conservative substitution of leucine (codon CTG) for proline (codonCCG) at residue 1114 of the amino acid sequence of the receptor (SEQ IDNO: 2). This P1114L mutation is predicted to lie in the intracellulartyrosine kinase domain II involved in intracellular signaling [Pajusolaet al., Cancer Res., 52:5738-5743 (1992)]. Direct sequencing ofpredicted exon 24 of the VEGFR-3 gene alleles from members of thisfamily identified this substitution only in affected and at-risk familymembers. This sequence change was not observed in 120 randomly selectedindividuals of mixed European ancestry from the general population (240alleles). In probands from the other 11 families, wild type sequence wasobserved at nucleotide position 3360.

Collectively, this data demonstrates that a missense mutation thatcauses a non-conservative substitution in a kinase domain of the VEGFR-3protein correlates strongly with a heritable lymphedema in one family,and suggests that other mutations in the same gene may exist thatcorrelate with heritable lymphedema in other families. As explainedabove, only a portion of the VEGFR-3 gene sequence was analyzed toidentify this first mutant of interest. Additional sequencing, usingstandard techniques and using the known VEGFR-3 gene sequence forguidance, is expected to identify additional mutations of interest thatare observed in affected and at-risk members of other families studied.

EXAMPLE 2

Demonstration that a C-T Missense Mutation at Position 3360 in theVEGFR-3 Coding Sequence Results in a Tyrosine Kinase Negative Mutant

The results set forth in Example 1 identified two missense mutations inthe VEGFR-3 coding sequence, one of which (C-T at position 3360)appeared to correlate with heritable lymphedema and one of which (C-Ttransition at position 1940) did not. The following experiments wereconducted to determine the biochemical significance of these mutationson VEGFR-3 biological activity.

To analyze how the two single amino acid substitutions affect theVEGFR-3-mediated signaling, the corresponding mutant receptor expressionvectors were generated using site-directed mutagenesis procedures andexpressed in 293T cells by transient transfection. The long form ofhuman VEGFR-3 cDNA (SEQ ID NO: 1) was cloned as a Hind III-Bam HIfragment from the LTR-FLT41 plasmid [Pajusola et al., Oncogene 8:2931-2937 (1993)] into pcDNA3.1/Z(+) (Invitrogen). The P641S and P1114Lmutants of VEGFR-3 were generated from this construct with theGeneEditor™ in vitro Site-Directed Mutagenesis System (Promega) usingthe following oligonucleotides (the C-T mutations are indicated withbold letters): (SEQ ID NO:17) 5′-CCTGAGTATC T CCCGCGTCGC-3′ for P641Smutation; and (SEQ ID NO:18) 5′-GGTGCCTCCC T GTACCCTGGG-3′ for P1114Lmutation.

For the transient expression studies, 293T cells were grown inDulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetalcalf serum (GIBCO BRL, Life Technologies, Gaithersburg, Md.), glutamine,and antibiotics. Cells were transfected with 20 μg of plasmid encodingthe wild type or mutant VEGFR-3 forms using the calcium phosphatemethod, and harvested 36 hours after transfection forimmunoprecipitation and Western blotting. Under these conditions, RTKoverexpression results in ligand-independent activation, thus allowingthe receptor phosphorylation to be studied. An empty vector was used formock (control) transfections. (It will be appreciated that ligandstimulation assays of VEGFR-3 forms also can be employed, e.g., asdescribed in U.S. Pat. No. 5,776,755, incorporated herein by reference,using VEGF-C or VEGF-D ligands.)

In order to investigate the effect of the two VEGFR-3 mutants on thetyrosine phosphorylation of the VEGFR-3, Western blotting analysis wasperformed using anti-phosphotyrosine antibodies. The cell monolayerswere washed three times with cold phosphate-buffered saline (PBS,containing 2 mM vanadate and 2 mM PMSF) and scraped into RIPA buffer(150 mM NaCl, 1% Nonidet P40, 0.5% deoxycholic acid sodium salt, 0.1%SDS, 50 mM Tris-HCl, pH 8.0) containing 2 mM Vanadate, 2 mM PMSF, and0.07 U/ml Aprotinin.

The cell lysates were sonicated and centrifuiged for 10 minutes at19,000×g, and the supernatants were incubated for 2 hours on ice with 2μg/ml of monoclonal anti-VEGFR-3 antibodies (9D9f9) [Jussila et al.,Cancer Res., 58: 1599-604 (1998)]. Thereafter, Protein A sepharose(Pharmacia) beads were added and incubation was continued for 45 minuteswith rotation at +4° C. The sepharose beads were then washed three timeswith ice-cold RIPA buffer and twice with PBS (both containing 2 mMvanadate, 2 mM PMSF), analyzed by 7.5% SDS-PAGE and transferred to anitrocellulose filter (Protran Nitrocellulose, Schleicher & Schuell, No.401196) using semi-dry transfer apparatus. After blocking the filterwith 5% BSA in TBS-T buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 0.05%Tween 20), the filters were incubated with the phosphotyrosine-specificprimary antibodies (Upstate Biotechnology, #05-321), followed bybiotinylated goat-anti-mouse immunoglobulins (Dako, E0433) andBiotin-Streptavidin HRP complex (Amersham, RPN1051). The bands werevisualized by the enhanced chemiluminescence (ECL) method.

After analysis for phosphotyrine-containing proteins, the filters werestripped by washing for 30 minutes at +50° C. in 100 mM2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7, with occasionalagitation. The filters were washed with TBS-T, blocked again with BSA asdescribed above, and analyzed for the presence of VEGFR-3 using the9D9f9 antibodies and HRP-conjugated rabbit-anti-mouse immunoglobulins(Dako, P0161).

The Western analyses revealed that the P641S mutant receptor wasphosphorylated normally, i.e., in a manner similar to the wild typecontrol. However, the proteolytic processing of the P641S receptorprotein may be affected, as the 175 kD and 125 kD polypeptides seemed tohave a higher relative density when compared to the 195 kD form.

In contrast, no phosphorylated P1114L mutant protein was detected usingthe phosphotyrosine antibodies. The expression of similar amounts of theVEGFR-3 protein (normal and both mutants) was confirmed using themonoclonal 9D9f9 antibody, which is directed towards the extracellulardomain of the VEGFR-3. Both the P641S and the P1114L mutant VEGFR-3migrated slightly faster than the wild type VEGFR-3 in the gelelectrophoresis.

In order to analyze the possible dominant negative effect of the P1114Lmutant on the wild-type receptor, a second, similar set of experimentswere performed wherein the 293T cells were transfected with anincreasing amount of the P1114L expression vector in combination withdecreasing amounts of the wild type vector. Wild type to mutant ratiosof 1:0, 3:1, 1:1, 1:3 and 0:1 were used. The cells were lysed 48 hoursafter transfection and the lysates were analyzed by immunoprecipitationand Western blotting as described above. These experiments permittedevaluation of whether the mutant protein interferes with wild typeprotein phosphorylation and estimation of the minimal amount of the WTprotein needed for observable tyrosyl autophosphorylation.Immunoprecipitates from cells transfected with only the WT plasmidrevealed WT protein that was strongly phosphorylated in this experiment(lane 2), whereas immunoprecipitates from cells transfected with onlythe mutant plasmid were again inactive (unphosphorylated).

Interestingly, when transfection was made using 75% of WT and 25% ofmutant plasmid, the phosphorylation of the receptors was decreased byabout 90%. This result strongly suggests that the P1114L mutant receptorforms heterodimers with the WT receptor, but cannot phosphorylate the WTreceptor, thus failing to activate it. Under this theory, the WTreceptor monomers in the heterodimers would also remain inactive,causing a disproportionate decrease of the total amount of activatedreceptor, when co-transfected with the mutant. Wildtype-wildtypehomodimers would remain active and be responsible for the observedsignaling. When the wild type and mutant receptor expression vectorswere transfected at a 1:1 ratio, the VEGFR-3 phosphorylation was about4% of the wild type alone, whereas at a 1:3 ratio, no tyrosinephosphorylation of VEGFR-3 was observed.

The foregoing results are consistent with the linkage analyses inExample 1: the mutation at position 641 that did not appear to correlatewith lymphedema also did not appear to be disfunctional, whereas themutation at position 1114 appeared to cause a dominant negative mutationthat shows no tyrosine phosphorylation alone and that drasticallyreduces VEGFR-3 signaling in cells expressing both the mutant and wildtype VEGFR-3 genes.

Collectively, these data indicate that the P1114L VEGFR-3 mutant isunable to act as a part of the signaling cascade, and also acts in adominant negative manner, thus possibly interfering partially with theactivation of the wild type VEGFR-3. Such effects of the mutation mayeventually lead to lymphedema.

EXAMPLE 3

Treatment of Lymphedema with a VEGFR-3 Ligand

The data from Examples 1 and 2 collectively indicate a causative role inheritable lymphedema for a mutation in the VEGFR-3 gene that interfereswith VEGFR-3 signaling. Such a mutation behaves in an autosomal dominantpattern, due to the apparent necessity for receptor dimerization in thesignaling process. However, the data from Example 2 suggests that someresidual signaling may still occur in heterozygous affected individuals,presumably through pairing of VEGFR-3 proteins expressed from the wildtype allele. The following experiments are designed to demonstrate theefficacy of VEGFR-3 ligand treatment in such affected individuals, toraise VEGFR-3 signaling to levels approaching normal and therebyameliorate/palliate the symptoms of hereditary lymphedema.

Initially, an appropriate animal model is selected. Several potentialanimal models have been described in the literature. [See, e.g., Lyon etal., Mouse News Lett. 71: 26 (1984), Mouse News Lett. 74: 96 (1986), andGenetic variants and strains of the laboratory mouse, 2nd ed., New York:Oxford University Press (1989), p 70 (Chylous ascites mouse); Dumont etal., Science, 282: 946-949 (1998) (heterozygous VEGFR-3 knockout mouse);Patterson et al., “Hereditary Lymphedema,”Comparative PathologyBulletin, 3: 2 (1971) (canine hereditary lymphedema model); van derPutte, “Congenital Hereditary Lymphedema in the Pig,” Lympho, 11: 1-9(1978); and Campbell-Beggs et al., “Chyloabdomen in a neonatal foal,”Veterinary Record, 137: 96-98 (1995),] Those models which are determinedto have analogous mutations to the VEGFR-3 gene are preferred. Analogousmutations would include mutations affecting corresponding residues andalso mutations affecting different residues but causing similarfunctional alterations. The Chylous ascites mouse VEGFR-3 gene containsa missense mutation at a position corresponding to residue 1053 of SEQID No. 2, which maps to the catalytic pocket region of the tyrosinekinase catalytic domain. Thus, the “Chy” mouse is expected to displaysimilar functional alterations to human mutations affecting tyrosinekinase activity, a prediction which can be confirmed by functionalassays such as those described in Example 2. In a preferred embodiment,“knock in” homologous recombination genetic engineering strategies areused to create an animal model (e.g., a mouse model) having a VEGFR-3allelic variation analogous to the human variations described herein.[See, e.g., Partanen et al., Genes & Development, 12: 2332-2344 (1998)(gene targeting to introduce mutations into another receptor protein(FGFR-1) in mice).] For example, the P1114L mutation in human VEGFR-3occurs in a VEGFR-3 region having highly conserved amino acid identitywith murine VEGFR-3 (Genbank Accession No. L07296). Thus, acorresponding P1114L can be introduced into the murine VEGFR-3 by“knock-in” homologous recombination. Optionally, such mice can be bredto the heterozygous VEGFR-3 knockout mice or Chy mice described above tofurther modify the phenotypic severity of the lymphedema disease.

The mice as described above are treated with a candidate therapeutic,e.g., a recombinant mature form of VEGF-C, at various dosing schedules,e.g., once daily by intravenous (IV) or intramuscular (IM) injection ata dose of 1-1000 ng/g body weight, preferably 10-100 ng/g, which shouldresult in a peak level saturating VEGFR-3 (K_(d) about 150 pM) but notVEGFR-2 (K_(d) around 400 pM). For VEGFR-3-specific forms, such asVEGF-CΔC₁₅₆, even higher dosing is contemplated, to sustainVEGFR-3-saturating physiological concentrations for longer periods.Direct IM injection at multiple sites in the muscles of affectedextremities is a preferred route of administration. The dosing isadjusted according to the efficacy of the treatment and the presence ofpossible side effects due to the lowering of blood pressure, which hasbeen observed in response to VEGF administration IV. The efficacy oftreatment is measured via NMRI imaging of the water content and volumeof swelling of the abdomen and the extremities of the animals. Theamount of fluid in the abdominal cavity is estimated and the animals areweighed during the follow-up.

In studies using VEGFR-3 −/+×Chy mice progeny, the animals will alsohave the β-galactosidase marker in their lymphatic endothelium. After asuccessful treatment, the treated and non-treated experimental animalsand VEGFR-3 −/+ controls are killed and their lymphatic vessels arevisualized by β-gal and antibody staining. The staining patterns ofexperimental and control animals are compared for vessel diameter,numbers of endothelial cells, density of blood and lymphatic vessels,and nuclear density/section surface area for the estimation of tissueoedema.

Such experiments are repeated with various candidate therapeutics (e.g.,VEGF-C or VEGF-D recombinant polypeptides; VEGF-C and VEGF-D genetherapy vectors; and combinations thereof) at various dosing schedulesto determine an optimum treatment regimen.

EXAMPLE 4

Chromosomal Structure of the Human VEGFR-3 Gene

Sequencing and mapping of human DNA corresponding to the VEGFR-3 locushas indicated that this gene consists of thirty exons separated bytwenty-nine introns of varying size. The exon intron organization issummarized as follows: EXON Bp of SEQ ID NO:1 NUMBER size (bp) INTRONSIZE 1  20-77 unknown  58 bp 2  78-174  >1 kb  97 bp 3  175-419  218 bp 245 bp 4  420-532  120 bp  113 bp 5  533-695  107 bp  163 bp 6  696-835 269 bp  140 bp 7  836-1004  261 bp  169 bp 8 1005-1122  >1 kb  118 bp 91123-1277 unknown  155 bp 10 1278-1440  >1 kb  163 bp 11 1441-1567unknown  127 bp 12 1568-1676 unknown  109 bp 13 1677-2039  293 bp  363bp 14 2040-2186  99 bp  147 bp 15 2187-2318 approx. 160 bp  132 bp 162319-2425  301 bp  107 bp 17 2426-2561 >464 bp  139 bp 18 2562-2666unknown  105 bp 19 2667-2780  143 bp  114 bp 20 2781-2869  >1 kb  89 bp21 2870-3020 unknown  151 bp 22 3021-3115 unknown  95 bp 23 3116-3238unknown  123 bp 24 3239-3350  974 bp  112 bp 25 3351-3450  400 bp  100bp 26 3451-3557 unknown  107 bp 27 3558-3705  >1 kb  148 bp 28 3706-3826unknown  121 bp 29 3827-3912 unknown  86 bp 30a (Flt4 short) 3913-4111 3.7 kb  199 bp 30b (Flt4 long) 3913-4416 (CDS 504 bp) >504 bp

The foregoing information permits rapid design of oligonudeotides foramplifying select portions of the VEGFR-3 gene from genomic DNA, or RNA,or cDNA, to facilitate rapid analysis of an individual's VEGFR-3 codingsequence, to determine whether the individual possesses a mutation thatcorrelates with a lymphedema phenotype

EXAMPLE 5

Identification of Additional Non-Consenative Missense Mutants

Using procedures essentially as described in Example 1, the VEGFR-3coding sequences from additional affected and unaffected individualsfrom families having members suffering from heritable lymphedema werestudied. The analysis focused on families with statistical linkage tochromosome 5q as described in Example 1. The additional analysisincluded the PCR amplification and sequencing of Exon 17, Exon 22, andExon 23 sequences with the following PCR primers: (SEQ ID NO:23) Exon17-1 5′-CATCAAGACGGGCTACCT-3′ (SEQ ID NO:24) Exon 17-25′-CCGCTGACCCCACACCTT-3′ (SEQ ID NO:25) Exon 22-15′-GAGTTGACCTCCCAAGGT-3′ (SEQ ID NO:26) Exon 22-25′-TCTCCTGGACAGGCAGTG-3′ (SEQ ID NO:27) Exon 23-15′-GAGTTGACCTCCCAAGGT-3′ (SEQ ID NO:28) Exon 23-25′-TCTCCTGGACAGGCAGTC-3′

These additional studies identified four additional non-conservativemissense mutations in evolutionarily conserved amino acids in kinasedomains I and II of human VEGFR-3. Each mutation, shown in Table 3below, was observed in a single independently ascertained family, and ineach family, the mutation co-segregates with individuals suffering from,or considered at risk for developing, lymphedema. None of thesemutations were observed in the VEGFR-3 genes in a random sample of morethan 300 chromosomes from individuals from families unafflicted withheritable lymphedema. TABLE 3 Mutations in VEGFR-3 causing HereditaryLymphedema* Nucleotide Amino Acid Functional Exon Substitution**Substitution Domain 24 C3360T P1114L Kinase 2 17 G2588A G857R Kinase 123 G3141C R1041P Kinase 2 23 T3150C L1044P Kinase 2 23 G3164A D1049NKinase 2*Numbers indicate nucleotide or amino acid positions in SEQ ID NOs: 1and 2.**It will be appeciated that, since DNA is double-stranded, eachmutation could be characterized in two equivalent ways, depending onwhether reference is being made to the coding or the non-coding strand.

Referring to SEQ ID NO: 2, the kinase domains of VEGFR-3 compriseapproximately residues 843-943 and residues 1009-1165. Within thesedomains, molecular modeling suggests that residues G852, G854, G857,K879, E896, H1035, D1037, N1042, D1055, F1056, G1057, E1084, D1096 andR1159 are of particular importance in comprising or shaping thecatalytic pocket within the kinase domains. See van Der Geer and Hunter,Ann. Res. Cell. Biol., 10: 251-337 (1994); and Mohammadi et al., Cell86: 577-587 (1996). Thus, this data identifying additional mutationsimplicate missense mutations within a kinase domain of the VEGFR-3protein as correlating strongly with a risk for developing a heritablelymphedema phenotype. Mutations which affect residues in and around thecatalytic pocket appear particularly likely to correlate withlymphedema. The P1114L mutation, though not situated within thecatalytic pocket, is postulated to cause a conformational alterationthat affects the catalytic pocket. The G857R mutation is postulated toblock the catalytic pocket and/or the ATP binding site of the kinasedomain.

EXAMPLE 6

Functional Analysis of Additional VEGFR-3 Missense Mutations

Using procedures essentially as described above in Example 2, thefunctional state of the G857R, L1044P, and D1049N mutations wereanalyzed. (PLCLB buffer, comprising 150 mm NaCl, 5% glycerol, 1% TritonX-100, 1.5M MgCl₂, 50 mm HEPES, pH 7.5, was substituted for RIPA bufferdescribed in Example 2 for immunoprecipitation and Western blottingprotocols.) A VEGER-3-encoding construct comprising the G857R mutationwas generated from the long form of human VEGFR-3 cDNA using theoligonucleotide: (SEQ ID NO:20) 5′-CGG CGC CTT CAG GAA GGT GGT-3′

A construct comprising the L1044P mutation was generated from the longform of human VEGFR-3 cDNA using the oligonucleotide: (SEQ ID NO:21)5′-CGG AAC ATT CCG CTG TCG GAA-3′

A construct comprising the D1049N mutation was generated from the longform of human VEGFR-3 cDNA using the oligonucleotide: (SEQ ID NO:22)5′-GTC GGA AAG CAA CGT GGT GAA-3′.

The constructs were transiently transfected into 293T cells andharvested for Western blotting essentially as described in Example 2,except for the buffer substitution described above. In contrast to wildtype VEGFR-3 and VEGFR-3 containing the P641S mutation, nophosphorylated G857R or L1044P mutant protein was detected using thephosphotyrosine antibodies, consistent with the results that had beenobserved for P1114L. The expression of similar amounts of the VEGFR-3protein was confirmed using the monoclonal 9D9f9 antibody, which isdirected towards the extracellular domain of the VEGFR-3 in the Westernblotting. This data suggested that these observed mutations did indeedaffect VEGFR-3 kinase function. The D1049N mutant appeared to retain atleast some tyrosine kinase activity. It is also noteworthy that VEGFR-1and VEGFR-2 contain an asparagine residue at the position in theirtyrosine kinase domains which corresponds to position 1049 of VEGR-3.Together, these data suggest that the D1049N variation may only be anallelic variant that correlates with hereditary lymphedema, rather thana causative mutation.

To determine whether the VEGFR-3 mutants function in a dominant negativemariner, each construct was co-transfected at varying ratios with wildtype receptor into 293T cells essentially as described in Example 2.Unlike the results observed for P1114L and described in Example 2,neither the G857R mutant nor the L1044P mutant seemed to interfere withphosphorylation of the co-transfected wild type receptor.

The absence of a dominant negative effect in these experiments does notforeclose a conclusion that the mutations described above are causative.It has been found that a significant fraction of ligand-activatedreceptor tyrosine kinases traffic to the lysosomal compartment afterinternalization, where they are degraded. However, receptors which arenot ligand-activated preferentially recycle back to the cell surfaceafter internalization. Thus, it is possible that the turnover time ofthe weakly phosphorylated mutant receptor is significantly longer thanthat of the wild type receptor protein. If this were true, the amount ofthe mutant receptor on the endothelial cell surface could beconsiderably higher than the amount of the phosphorylated and rapidlyinternalized wild type receptor, and any available ligand would thusbind a disproportionally high number of mutant receptors. Both apossible dominant negative effect of the mutant receptor and anabnormally long half-life of the tyrosine kinase negative mutantreceptor could eventually lead to lymphedema. Alternatively, a mutationthat merely decreases (but does not eliminate) VFGFR-3 tyrosine kinaseactivity may display a constitutive low level of internalization anddegradation that is insufficient to trigger sufficient downstreamsignalling, but decreases the effective concentration of VEGFR-3 on cellsurfaces for ligand binding and effective activation, leading eventuallyto lymphedema.

While the present invention has been described in terms of specificembodiments, it is understood that variations and modifications willoccur to those in the art, all of which are intended as aspects of thepresent invention. Accordingly, only such limitations as appear in theclaims should be placed on the invention.

1-11. (canceled)
 12. A method of treatment for hereditary lymphedema,comprising: administering to a patient with hereditary lymphedema atherapeutically effective amount of a growth factor product selectedfrom the group consisting of vascular endothelial growth factor C(VEGF-C) protein products, vascular endothelial growth factor D (VEGF-D)protein products, and VEGF-D gene therapy protein products, wherein saidpatient with hereditary lymphedema comprises a mutation that alters theencoded amino acid sequence of at least one VEGFR-3 allele of thepatient, wherein said mutation reduces ligand-mediated signaling of theVEGFR-3 polypeptide encoded by the allele, when compared to VEGFR-3encoded by a wild-type human VEGFR-allele; and wherein saidtherapeutically effective amount of said growth factor product isadministered locally at a site of edema in the patient.
 13. Atherapeutic or prophylactic method of treating lymphedema, comprisingthe steps of: providing isolated lymphatic endothelial cells orlymphatic endothial progenitor cells; transforming or transfecting thecells ex vivo with a polynucleotide comprising a nucleotide sequencethat encodes a wild type VEGFR-3; and administering the transformed ortransfected cells to the mammalian subject. 14-21. (canceled)
 22. Apurified polynucleotide comprising a nucleotide sequence encoding ahuman VEGFR-3 protein variant, wherein said polynucleotide is capable ofhybridizing to the complement of SEQ ID NO: 1 under the followinghybridization conditions: hybridization at 42° C. in 50% formamide,5×SSC, 20 mM Na·PO₄, pH 6.8; and washing in 0.2×SSC at 55° C.; andwherein the encoded VEGFR-3 protein variant has an amino acid sequencethat differs from the amino acid sequence set forth in SEQ ID NO:2 atone or more positions selected from the group consisting of amino acids843 to 943 of SEQ ID NO: 2 and amino acids 1009 to 1165 SEQ ID NO: 2.23-29. (canceled)
 30. A method for identifying a modulator ofintracellular VEGFR-3 signaling, comprising the steps of: a) contactinga cell expressing at least one mutant mammalian VEGFR-3 polypeptide inthe presence and in the absence of a putative modulator compound; b)detecting VEGFR-3 signaling in the cell; and c) identifying a putativemodulator compound in view of decreased or increased signaling in thepresence of the putative modulator, as compared to signaling in theabsence of the putative modulator. 31-36. (canceled)
 42. The method ofclaim 12 or 37, wherein the wildtype VEGFR-3 allele comprises theVEGFR-3 coding sequence set forth in SEQ ID NO:
 1. 43. The method ofclaim 12 or 37, wherein said administering of said therapeuticallyeffective amount of said growth factor product induces VEGF-3 signalingin the lymphatic endothelia of the patient.
 44. The method of claim 12or 37, wherein said administering of said therapeutically effectiveamount of said growth factor product reduces edema in a limb of saidpatient.
 45. The method of claim 12 or 37, wherein said administering ofsaid therapeutically effective amount of said growth factor productreduces accumulation of lymph fluids in said patient.
 46. The method ofclaim 45, wherein the administering step comprises administering acomposition that comprises the growth factor product and apharmaceutically acceptable carrier.
 47. The method of claim 46, whereinsaid growth factor product comprises a VEGF-D protein product comprisinga member selected from the group consisting of: (a) a polypeptidecomprising the amino acid sequence of SEQ ID NO: 6; (b) a polypeptidethat comprises an amino acid sequence that comprises a continuousportion of SEQ ID NO: 6 sufficient to permit the polypeptide to bindwildtype human VEGFR-3 and stimulate VEGFR-3 phosphorylation in cellsthat express wildtype human VEGFR-3; (c) a polypeptide of (a) or (b),with the proviso that the polypeptide has up to 25 amino acids added,deleted, or substituted with another amino acid, compared to the aminoacid sequence of the polypeptide of (a) or (b), and wherein thepolypeptide retains the ability to bind and stimulate phosphorylation ofwildtype human VEGFR-3.
 48. The method of claim 47, wherein the VEGF-Dprotein product comprises amino acids 93-201 of SEQ ID NO:
 6. 49. Themethod of claim 47, wherein said growth factor product is administeredvia intravenous injection.
 50. The method of claim 47, wherein saidgrowth factor product is administered via intramuscular injection.